LOCUS       Exported                8804 bp ds-DNA     circular SYN 03-AUG-2016
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    mpCAR-pNIC28
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8804)
  AUTHORS   Will
  TITLE     Direct Submission
  JOURNAL   Exported 23 Sep 2016 from SnapGene 2.6.2
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8804
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..19
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) recognition and cleavage
                     site"
                     /note="TEV site"
                     /translation="ENLYFQS"
     CDS             155..3643
                     /codon_start=1
                     /note="mpCAR"
                     /translation="MASESRDVRLQRRIAELYDTDPQFAAARPDEAVARAVNAPGLTLS
                     QVIRTVLDNYADRPALGYRAVEFAADPASARTVARLLPRFDTITYRELGERIDATTAAL
                     GHDGLRPGERVAILGFSSVDYTTIDIAAFNLGAVSVPLQTSAPPSQLRPMTAETEPAVI
                     AASVDFLDDALELIRTGHAPRRLVVFDFHPEIDDHRDALAAATTGLADTAMTVETLDSL
                     LTRGRTLPAPTGYRDRDDDLALLIYTSGSTGAPKGAMYQRRMVTNMWRRATTAIWGGKE
                     AAPWLTLNFMPMSHVMGRGILSTTLCSGGTAYFAARSDLSTLLEDLALVRPTQLTFVPR
                     IWEMIFQEYQRDVARRPEAEVLADLREHRLGGRFLAAMTGSAPMSPEMTAFAESVLDLH
                     LVDGYGSTEAGSIVLDGQVLRPPVLDYKLVDVPELGYFSTDRPYPRGELLVKTELMFPG
                     YYKRPDITAEVFDEDGYYRTGDIVAELGPDRLAYVDRRNNVLKLSQGEFVTVSKLEAAF
                     AASPLVRQIYIYGNSAHPYLLAVVVPTEDALTRYDAATLKTAISESLQDVGRAAGLQSY
                     EIPRDFLVETTPFTLENGLLTGLRKLARPKLKEHYGERLEQLYTELADGQAEELKELRT
                     HGAQQPTLTTVSRAATALLGTASAEVRPDAHFTDLGGDSLSALTFGNLLGEIYAVEVPV
                     GVIVSPANDLAAIADYIDTARRPGDGRPTFAGVHGDDAAEVHARDLTLDRFLDDATLAA
                     ATALPGPAPEIRTVLLTGATGFLGRYLALEWLERMAMVGGTLICLVRGRDDAAARARLD
                     QIFDSGDPELLRHYRELADRHLEVIAGDKSDADLGLDRRTWQRLADTVDLIVDPAALVN
                     HVLPYRELFGPNVVGTAELIRLALTGRLKPYLYTSTIAVGAGIAPGQFTEDADIRQISA
                     TRTLDDSYANGYATSKWAGEVLLREAHDLCGLPVAVFRCDMILADTSYAGQLNVPDMFT
                     RLILSLVATGIAPLSFYELDAAGHRQRAHYDGLPVEFVAEAVSALGLDVAEDGGFATYH
                     VMNPYDDGIGLDEFVDWLTGAGYPIEHVNDYGTWFQRFETAIRGLPERQRQASLLPLLH
                     SYQRPQPPIRGSAAPTDRFRSAVQDAKIGPDKDIPHITPEVIVKYVTDLRLLGLL"
     CDS             3665..3682
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     terminator      3749..3796
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3833..4288
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(4381..5196)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /note="KanR"
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      5318..5906
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    6092..6234
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     CDS             complement(6336..6527)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7336..8418)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(8419..8496)
                     /gene="lacI"
                     /note="lacI promoter"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatggca tcagaatccc gtgacgttcg
      181 cctgcagcgc cggatcgccg agttgtacga caccgatccg cagttcgccg cggcgcgccc
      241 cgacgaggcc gtcgcacggg cggtcaacgc accggggctg acgctgtcgc aggtcatccg
      301 gaccgtgctg gacaactacg ccgaccgtcc cgccctcggc taccgcgccg tcgagttcgc
      361 cgccgacccc gcctccgccc gcacggtggc gcgactactc ccccgcttcg acaccatcac
      421 ctaccgcgag ctcggcgaac ggatcgacgc gaccaccgcc gcgctgggcc acgacggcct
      481 gcgccccggc gagcgcgtcg cgatcctggg tttctccagc gtcgactaca ccaccatcga
      541 catcgcggcg ttcaatctcg gcgcggtgtc ggtgcccctg cagaccagcg cgccgccctc
      601 ccaactgcgc ccgatgaccg ccgagaccga accggccgtc atcgccgcct cggtcgactt
      661 cctcgatgac gcgctcgaac tcatccgcac cggccacgcg ccgcggcggc tggtcgtgtt
      721 cgacttccac cccgagatcg acgaccaccg cgacgccctg gccgccgcga cgaccggact
      781 ggccgacacg gcgatgacgg tggagaccct cgactccctg ctgacccgcg ggcgcaccct
      841 gcccgcgccg accggctacc gggaccgcga cgacgacctc gcgctgctca tctacacctc
      901 cggcagcacc ggcgcgccga aaggtgcgat gtaccaacgg cggatggtca ccaacatgtg
      961 gcggcgcgcc accaccgcga tctggggcgg gaaggaggcg gcgccgtggc tgacgctgaa
     1021 cttcatgccg atgagccacg tgatgggccg cggtatcctc agcaccacac tgtgctccgg
     1081 cggcaccgcg tatttcgctg cgcgaagcga cctttcgaca ctgctggagg atctggcgct
     1141 ggtccggccg acacagctga ccttcgtgcc gcgcatctgg gagatgatct tccaggagta
     1201 tcagcgcgac gtggcccgcc ggcccgaggc cgaggtgctg gccgacctgc gcgagcaccg
     1261 cctcggcggg cggttcctcg ccgcgatgac cggatccgcg cccatgtcac cggagatgac
     1321 ggcgttcgcc gagtccgtgc tcgatctgca cctcgtcgac gggtacggat ccaccgaggc
     1381 cggcagcatc gtgctcgacg ggcaggtgct gcgcccgccg gtgctcgact acaagctggt
     1441 cgacgtgccc gagttgggct acttcagcac cgaccggccc tatccgcgcg gggagctgct
     1501 ggtcaagacc gagctgatgt ttccgggcta ctacaagcgg ccggacatca ccgcggaggt
     1561 gttcgacgag gacggctact accggaccgg cgacatcgtc gccgaactcg gtcccgaccg
     1621 gctggcctac gtcgaccggc gcaacaacgt gctcaagctc tcccagggcg agttcgtcac
     1681 cgtctccaag ctggaggccg cgttcgccgc cagcccgctg gtgcggcaga tctacatcta
     1741 cggcaacagc gcccacccgt acctgctcgc cgtcgtcgtc cccaccgagg acgcactgac
     1801 ccgctacgac gcggcgacgc tgaaaaccgc gatcagcgag tcgcttcagg acgtcggccg
     1861 cgccgccggc ctgcagtcct acgagatccc acgcgacttc ctggtcgaga caacaccgtt
     1921 cacgctggag aacggtctgc tcaccgggct ccgcaagctc gcgcggccga aactcaagga
     1981 gcactacggc gaacgcctcg aacagctcta caccgaactg gccgacggtc aggccgagga
     2041 gctgaaggag ctgcgcacac acggcgcgca gcagcccacc ctgacgaccg tcagccgcgc
     2101 cgcgaccgca ctgctgggca ccgcgtccgc cgaggtgcga cccgacgcgc acttcaccga
     2161 cctgggcgga gactcgttgt cggcgttgac gttcggcaac ctgctcgggg agatctacgc
     2221 cgtcgaggtg cccgtcggcg tgatcgtcag ccccgccaac gacctcgccg cgatcgccga
     2281 ctacatcgac accgcgcgcc gaccaggcga cggacggccc accttcgccg gggtccacgg
     2341 cgacgacgcc gccgaggtgc acgcccgcga tctgaccctg gacaggttcc tcgacgacgc
     2401 gacgctggcc gccgcgaccg ctctgcccgg tccggcgccg gagatccgca cggtgctgct
     2461 gaccggggcg accggattcc tcggccgcta cctggcgctg gagtggctgg agcggatggc
     2521 gatggtcggc ggcaccctga tctgcctggt gcgcggccgc gacgacgccg ccgcccgggc
     2581 ccggctggat cagatcttcg actccggcga tcccgaactg ctgcgccact accgggagtt
     2641 ggccgaccgc catctcgagg tcatcgccgg cgacaagagc gacgccgacc tcggcctgga
     2701 ccgccggacc tggcagcggc tggccgatac cgtcgacctg atcgtcgacc ccgccgccct
     2761 ggtcaaccat gtgctgccgt accgggagtt gttcgggccc aacgtggtcg gaaccgccga
     2821 gctgatccgt ctcgcgctca ccggcaggct caagccgtat ctgtacacct cgacgatcgc
     2881 tgtgggcgcg gggattgcgc ccgggcagtt caccgaggac gccgacatcc ggcagatcag
     2941 cgccacccgg acactcgatg acagctacgc caacggctac gccaccagca agtgggccgg
     3001 cgaggtgctg ctgcgggagg cacacgatct gtgcggcctg ccggtggcgg tgttccggtg
     3061 cgacatgatc ctggccgaca ccagctacgc cggtcagctc aacgtgccgg acatgttcac
     3121 ccggctgatc ctgagcctgg tggccaccgg gatcgcgccg ctgtcgttct acgaactcga
     3181 cgccgccggg caccggcagc gggcgcacta cgacggtctg ccggtggagt tcgtcgccga
     3241 ggccgtctcc gcactcggtc tcgacgtggc cgaggacggc ggcttcgcga cctaccacgt
     3301 gatgaacccg tacgacgacg gcatcgggct cgacgagttc gtcgactggc tgaccggggc
     3361 cgggtatccg atcgagcacg tcaacgacta cggcacctgg tttcagcggt tcgagaccgc
     3421 gatccgcggc ctgcccgaac ggcagcggca ggcgtcgctg cttccgctgc tgcacagcta
     3481 ccagcggccg cagccgccga tccgcgggtc ggccgccccg accgatcggt tccgcagcgc
     3541 ggtgcaggac gcgaagatcg gcccggacaa ggacattccg cacatcaccc cggaggtgat
     3601 cgtcaagtac gtcaccgatc tgcggctgct cgggctgctc tgaaagcttg cggccgcact
     3661 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     3721 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     3781 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     3841 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3901 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3961 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4021 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     4081 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4141 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     4201 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4261 tttaacaaaa tattaacgtt tacaatttca ggtggcactt ttcggggaaa tgtgcgcgga
     4321 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     4381 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     4441 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     4501 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     4561 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     4621 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     4681 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     4741 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     4801 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     4861 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     4921 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     4981 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     5041 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     5101 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     5161 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     5221 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     5281 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5341 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     5401 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     5461 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     5521 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     5581 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5641 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     5701 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     5761 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     5821 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     5881 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5941 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     6001 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     6061 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     6121 tgtgcggtat ttcacaccgc atatatggtg cactctcagt acaatctgct ctgatgccgc
     6181 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     6241 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     6301 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     6361 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     6421 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     6481 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     6541 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     6601 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     6661 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     6721 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     6781 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     6841 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     6901 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     6961 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     7021 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     7081 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     7141 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     7201 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     7261 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     7321 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     7381 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     7441 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     7501 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     7561 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     7621 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     7681 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     7741 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     7801 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     7861 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     7921 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     7981 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     8041 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     8101 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     8161 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     8221 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     8281 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     8341 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     8401 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     8461 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     8521 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     8581 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     8641 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     8701 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     8761 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaat
//
