LOCUS       sequence-333539-        8771 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AncCAR_A in E. coli from a T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8771)
  AUTHORS   Thomas A, Cutlan R, Finnigan W, van der Giezen M, Harmer N
  TITLE     Highly thermostable carboxylic acid reductases generated by 
            ancestral sequence reconstruction.
  JOURNAL   Commun Biol. 2019 Nov 22;2:429. doi: 10.1038/s42003-019-0677-y. 
            eCollection 2019.
  PUBMED    31799431
REFERENCE   2  (bases 1 to 8771)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Commun 
            Biol."; date: "2019-11-22"; volume: "2:429. doi"; pages: " 
            10.1038/s42003-019-0677-y. eCollection 2019"
COMMENT     Sequence Label: pNIC-AncCAR_A
FEATURES             Location/Qualifiers
     source          1..8771
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3636..3653
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3706..3724)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3720..3767
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3804..4259
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3891..3910)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4101..4122
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4351..5166)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4351..5166)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5074..5093
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5158..5177
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5288..5876
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5777..5796
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6030..6047
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6062..6201
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6127..6146)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6287..6309
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6303..6494)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7303..8385)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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"
     primer_bind     8347..8366
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8386..8463)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8669..8688
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatgagc gtagataccc gtgaggagcg
      181 cctggaacgt cgcatcgccg agctttacgc aaccgatccc caatttcgtg cggcgatgcc
      241 tgacgaagct gtcaccgagg cggtccgtca gccggggctg cgtttagctc aaattttaga
      301 gaccctgatg gaaggttatg cagatcgtcc agcgttggga cagcgtgcta ccgaactgac
      361 aggccgcacc acgccttttg aaaccattac gtacggggag ttatgggaac gtgtcggtgc
      421 gattgctgcg gcctggcatc atgaccctgt tcgtcccggc gattttgtcg cgatccttgg
      481 gttcacttcc cctgactata caaccctgga tcttgcatgt atccacttag gagcagtctc
      541 cgtgccgctt caagcatccg catcggtggc acagctgcgt cctattcttg aagaaactga
      601 gccacgtgta ctggccgcgt ccgcggagca tcttgatgcg gctgtagagt gtgtattgtc
      661 cagtagttcg cctcgtcgtc ttgtcgtctt cgattatcat cccgaagtgg atgaccaccg
      721 tgaagcatta gagtccgctc gtgctcgctt ggcggaagca ggaagcccgg tcacggtgga
      781 gacgttagac gaagtaattg aacgcggacg tgccctgcct gctcccccgc tgtttactga
      841 ccccgacgat gatcctctgg ctttacttat ttatacctca gggtcgactg gcacaccgaa
      901 aggagcaatg tacacggaac gtttggtagc tcgtttctgg ttatcttcat catcacctgg
      961 ggagggggaa gttccgtcga tcaacttgaa ctatatgccg atgagccata tcgcggggcg
     1021 cgccagcttg tatggtacac tggcgagcgg gggcactgcg tatttcgctg ccaagtctga
     1081 tttgtcgaca ttgttcgagg atatcgcctt ggtccgccca actgagttag gtctggtgcc
     1141 gcgtgtttgc gacatgttat ttcaacgcta tcaatccgcc gtagacgcgg atcgtgaaac
     1201 tttagaagct gaggcgaaga ccgagttgcg cgagcatgta ttaggcggcc gttttttaac
     1261 tgcaatgtgc ggctccgctc ccctttctgc cgaaatgaaa gcattcatgg agtccgtatt
     1321 agatttacat cttttggatg gctatggaag taccgaagcc ggttctgtac ttcgtgacgg
     1381 caaaattcaa cgcccgccgg taattgacta caaactggtt gatgttccgg agttaggtta
     1441 cttccgtacc gataaaccgc acccccgtgg ggagctgtta gtcaaaagcg agacaatgat
     1501 tcctgggtat tataaacgcc ccgaagtgac tgcggagatt tttgatgaag atggatttta
     1561 ccgcaccggt gacgtaatgg ctgagatcgg cccagaccgt ctggtgtacg ttgaccgccg
     1621 caacaatgtc ctgaagctgt cccagggcga gttcgtggca gtagcgaaat tggaggcggt
     1681 ctttgcgggt tccccgttag tacgccaaat cttcgtgtac gggaactctg agcgctccta
     1741 cttgttagca gtggttgttc cgactgaaga cgcccttgct cgccccgacc cggaggaatt
     1801 aaaacccgcc atcagcgaga gtcttcagca gatcgccaag gaagcggggt tgcagagtta
     1861 cgaaattccg cgcgatttta ttattgaaac agagcccttc acaattgaga acgggctgtt
     1921 gtccggtatt ggaaaacttc tgcgtccgaa gttaaaggaa cgttacggag agcgcttaga
     1981 gcagctgtac gccgagttgg ccgaaggtca ggctgacgaa cttcgtgcgc ttcgcgagtc
     2041 ggcagcagat cgccctgtac ttgaaacagt gagccgtgca gcgaaagcat tgcttggcgc
     2101 cgccgcggcc gacctgcgtc ctgatgcaca cttcacggat ctgggtggcg atagtctttc
     2161 tgctctgtcg ttttcgaatc tgttacgtga tatctttgat gttgaggtac cggttggggt
     2221 aatcgttggg ccggccaata atttacgcca gttggctgag tatatcgaag cagaacgcgc
     2281 ctcaggctct cgtcgcccaa ccttcgcctc cgtccacgga gcgggcgcaa cggaaatccg
     2341 tgccgctgat ctgactttgg ataagttcat tgatgctgag actttagccg cagccccctc
     2401 tttaccacgt ccgtccggga ccccccgcac agtgttgttg acaggggcaa atggatactt
     2461 aggacgcttc ctgtgtttgg agtggttgca acgtcttgat gagaccggtg gcaaactgat
     2521 ctgtctggtc cgtggctctg atgcggaggc tgcacgcgcc cgtctggaag aggcttttga
     2581 tagcggcgat ccagaactgc tggagcactt tcgtgagttg gcggctgaac accttgaagt
     2641 aatcgcaggt gacattgggg agccaaatct ggggctggac gaggcaacgt ggcaacgtct
     2701 tgcagaaacg gtcgatctta ttgttcaccc cgcggctttg gttaatcatg tattacctta
     2761 tgaccaactg ttcggcccaa atgtagttgg tactgctgaa attattcgct tagcattgac
     2821 cactcgcctt aagcccgtga cgtatctgtc aactgttgcc gttgcggctc aggtcgaccc
     2881 agctgcattt gaagaagatg gagatatccg cgagatttct ccggtacgcc caatcgacga
     2941 cagctacgca aacgggtatg gtaattcgaa gtgggcaggc gaggttcttc tgcgtgaggc
     3001 acatgatttg tgtggtttac cagttgccgt ctttcgttcc gacatgattc ttgcgcattc
     3061 gcgttacgcg ggtcagttaa acgtaccaga tatgtttact cgcttacttc tgtccttgtt
     3121 ggccaccggt attgccccta agagttttta tcaaaccgat gccgacggaa accgccagcg
     3181 cgcgcactac gatggactgc cggtagattt caccgcggaa gcaatcacta ctttgggggc
     3241 tcaggcaacg gacggttacc gtacgtatga cgtcatgaat ccacacgacg atgggatcag
     3301 cttagacgaa ttcgtagact ggttaattga agctggacat ccaatcgaac gcattgatga
     3361 ctatgacgag tggtttgccc gcttcgaaac ggcgttgcgt gctttacctg aaaagcaacg
     3421 ccagcattcc gtcctgccgt tgctgcacgc gtatcgccgt cctgcaccac cgattcgtgg
     3481 atcagcgtta ccggccgaac gttttcgcgc ggcggttcaa gaggccaaaa tcggcccgga
     3541 caaggatatc ccccatcttt ctccggaatt gattgagaaa tatgtgaccg accttcgcct
     3601 tctgggtctt ctttaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc
     3661 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     3721 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     3781 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3841 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3901 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3961 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4021 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4081 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4141 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4201 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     4261 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     4321 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     4381 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     4441 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     4501 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     4561 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     4621 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     4681 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     4741 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     4801 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     4861 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     4921 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     4981 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     5041 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     5101 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     5161 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     5221 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5281 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5341 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5401 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     5461 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5521 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5581 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5641 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5701 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5761 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5821 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5881 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     5941 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     6001 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     6061 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     6121 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     6181 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     6241 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     6301 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     6361 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     6421 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     6481 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     6541 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     6601 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     6661 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     6721 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     6781 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     6841 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     6901 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     6961 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     7021 gtgacgaagg tttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     7081 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     7141 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     7201 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     7261 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     7321 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     7381 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     7441 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     7501 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     7561 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     7621 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     7681 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     7741 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     7801 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     7861 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     7921 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     7981 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     8041 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     8101 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     8161 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     8221 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     8281 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     8341 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     8401 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     8461 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     8521 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     8581 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     8641 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     8701 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     8761 tcccgcgaaa t
//
