LOCUS       sequence-425842-        6647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for TtA gene, codon optimized for expression in
            E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6647)
  AUTHORS   Bell EL, Rosetto G, Ingraham MA, Ramirez KJ, Lincoln C, Clarke RW, 
            Gado JE, Lilly JL, Kucharzyk KH, Erickson E, Beckham GT
  TITLE     Natural diversity screening, assay development, and characterization
            of nylon-6 enzymatic depolymerization.
  JOURNAL   Nat Commun. 2024 Feb 9;15(1):1217. doi: 10.1038/s41467-024-45523-5.
  PUBMED    38336849
REFERENCE   2  (bases 1 to 6647)
  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: "Nat 
            Commun."; date: "2024-02-9"; volume: "15(1):1217. doi"; pages: " 
            10.1038/s41467-024-45523-5"
COMMENT     Sequence Label: TtA_pET29b
FEATURES             Location/Qualifiers
     source          1..6647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1417..1434
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1487..1505)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1501..1548
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1585..2040
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1672..1691)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1882..1903
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2132..2947)
                     /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(2132..2947)
                     /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     2855..2874
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2939..2958
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3069..3657
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3558..3577
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3811..3828
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3843..3982
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3908..3927)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4068..4090
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4084..4275)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5084..6166)
                     /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     6128..6147
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6167..6244)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6450..6469
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6557..6576
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6557..6575
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6576..6600
                     /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             6615..6637
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 cacatccatg agtacacgcg ttacgatgct gtgggcctgc gtaacctgat aaaaagcgga
       61 gaggtgacgg cggccgaagt cgagaccgct gcccgtgagg ccttagcagc cgccgatgaa
      121 cgcgtcaatg gtctcgcgct gccattattc actccggcac tggggcacgc agaagacggg
      181 ccattcgctg gtgtcccctt cctgatcaaa gatattggcc ctatggctgc tggtgtaccc
      241 ttctttatgg gatcgcgtgc attaggcccc ggggtgcccg ctgctcacga tagcgatctg
      301 atgcgccgtt tccgcgctgc gggattggtc acgttgggtt tgacgacaat gccagaactg
      361 gggctctgct ttgcaacgga gcctgtacgt tttggaccag ctcgtaatcc ctgggacctg
      421 gatcgggggg caggggggtc ttccggcgga gctgccgccc tggtagcggc aggcgctgtt
      481 ccattggcgc acgcctctga cggcgccgga tctatccgca ttcccgccgc gtgttgcggc
      541 ttagtagggc ttaaaccctc acgtggacgc cttccctgcg gtcctgacct tggtgaaagt
      601 gcactgggca tggcggacgc cttcgctctg actcgtacag ttcgagatac cgctcatttg
      661 cttgatgcag ttcagggacc tggtgtgggc gagaaatata ccgcacctcc gccggcgcgt
      721 ccgtatgccg aagaacctac acgcgaaccg cggcgccttc gtgtagccat aacaaccaaa
      781 ccctggtctg atgcaccagt aaatccagaa gtagccgcag ctgcagagca tgccgctcgt
      841 ttactgcagc agcaaggcca ccatgttgaa gaggcatcgc cggttgttga ttgggatgcg
      901 gtcattcatg cgttagtggt aacccaaacg gcctttctgg gtgcaatttt tcagcgtgcg
      961 ccgcgtcccc ccgacccagc ccttttggag ggcgtgtctc gccgagtttt ggaacaggcc
     1021 gccagactct ctgctttaga tctgctggca gcatttgatg cgcagaatcg cgttactcga
     1081 agcgtcgccg cgttctttac tcgttatgat ctgctggtat ctccgacaat ggctcagtta
     1141 ccagctccac atggtactat ccgctatgac gatccttctc atactttaga aacctttgtg
     1201 agatctatgt ttgattatgc cccatttacg ccaccgttca atgtgacggg tcagcccgca
     1261 atttcactgc cactggggcg gtccactggt ggtttaccta tcggcgtcca actggtggcc
     1321 gcatatggtc gcgaagatct tctgctggga attgccggta gactggagca ggcagctccg
     1381 tggcgtagac acgcggatac tctgtttcgt ctcgagcacc accaccacca ccactgagat
     1441 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     1501 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     1561 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1621 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1681 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1741 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1801 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1861 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1921 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1981 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2041 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2101 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     2161 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     2221 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     2281 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     2341 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     2401 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     2461 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     2521 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     2581 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     2641 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     2701 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     2761 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     2821 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     2881 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     2941 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     3001 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3061 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3121 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3181 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3241 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3301 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3361 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3421 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3481 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3541 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3601 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3661 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3721 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3781 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3841 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     3901 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     3961 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4021 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4081 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4141 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     4201 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     4261 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     4321 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     4381 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4441 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     4501 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4561 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4621 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4681 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     4741 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     4801 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     4861 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     4921 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     4981 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5041 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5101 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5161 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     5221 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     5281 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     5341 tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg
     5401 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     5461 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     5521 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     5581 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     5641 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     5701 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     5761 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     5821 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     5881 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     5941 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     6001 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     6061 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     6121 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     6181 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     6241 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     6301 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     6361 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     6421 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     6481 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat
     6541 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     6601 cctctagaaa taattttgtt taactttaag aaggagatat acatatg
//
