LOCUS       sequence-326282-        8775 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 8775)
  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 8775)
  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..8775
                     /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(4352..5167)
                     /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(4352..5167)
                     /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     5075..5094
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5159..5178
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5289..5877
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5778..5797
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6031..6048
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6063..6205
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6131..6150)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6291..6313
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6307..6498)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7307..8389)
                     /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     8351..8370
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8390..8467)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8673..8692
                     /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 atattaacgt ttacaatttc
     4261 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4321 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     4381 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     4441 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     4501 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     4561 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     4621 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     4681 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     4741 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     4801 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     4861 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     4921 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     4981 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     5041 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     5101 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     5161 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     5221 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5281 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5341 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5401 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     5461 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5521 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5581 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5641 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5701 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5761 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5821 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5881 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5941 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     6001 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     6061 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatatggt
     6121 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     6181 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     6241 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     6301 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     6361 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     6421 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     6481 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     6541 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     6601 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     6661 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     6721 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     6781 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     6841 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     6901 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     6961 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     7021 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     7081 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     7141 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     7201 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     7261 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     7321 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     7381 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     7441 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     7501 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     7561 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     7621 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     7681 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     7741 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     7801 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     7861 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     7921 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     7981 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     8041 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     8101 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     8161 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     8221 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     8281 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     8341 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     8401 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     8461 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     8521 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     8581 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     8641 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     8701 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     8761 tcgatcccgc gaaat
//
