LOCUS       sequence-426050-        6704 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression construct for GatA gene, codon optimized for expression 
            in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6704)
  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 6704)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: GatA_pET29b
FEATURES             Location/Qualifiers
     source          1..6704
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1474..1491
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1544..1562)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1558..1605
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1642..2097
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1729..1748)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1939..1960
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2189..3004)
                     /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(2189..3004)
                     /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     2912..2931
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2996..3015
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3126..3714
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3615..3634
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3868..3885
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3900..4039
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3965..3984)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4125..4147
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4141..4332)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5141..6223)
                     /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     6185..6204
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6224..6301)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6507..6526
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6614..6633
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6614..6632
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6633..6657
                     /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             6672..6694
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 acaggtctgc acttcagatc cgcgtccgaa ctgggtcgaa tgataagacg cggagaaata
       61 tcttcagctg agttgacaga tcattttatc cagagaatag agacactcga tggtaaaacg
      121 aatgccgtgg ttgcccggga tttcgatcgt gcacgcgcac tggccaagga agctgacgca
      181 gcacaggccc gcggcgcttc tttaggtgcc ctgcacggac tgccgtttac aattaaggat
      241 gcctatgaag tggaaggtat cgtatctaca ggtggaaatc caacttggaa agatcacgtg
      301 ccaacgtcat cagcaaccgc cgtcgaacgt cttcagcggt ctggcgctat tgtcatgggt
      361 aaaacgaacg tcccatatct ttctggtgat ctgcagactt ataatgacat atatggtact
      421 acaaacaacc cctgggcact tgactgcggt cctgggggta gctcaggcgg gagcgcagct
      481 tctctggcgg ccggttttgc tgcggccgaa tttggttctg acatcggagg cagcatccgc
      541 actccggccc atctctgcgg agtatttggc cacaagcctt cgttcggtat cgttcctaaa
      601 cgcggtcatc tctctccgcc cccgggatgc ttaagcgaag gcgatttatc tgtggcagga
      661 ccacttgcac gcagcgcaga ggatctgaag ctgttactga gcctcactgc agggccggat
      721 tgggcggatg ccgccggctg gaaactcgat ttaccgccag cacgcgcccg tactcctcgg
      781 gagctgcggg cggcagtctg gattgatgac gaattctgcg acattgatcg cgaatcagct
      841 gacctcctgc gtaatgccgc aaaagcactg caggatgccg gagccaacgt tgattggaat
      901 gcccgacctg atttcaccct tgctgaaata actgagtgtt atctgatcct cctgcactct
      961 caaataggcg caggcatgcc acagtcaatc agagaccatt gggctgaaat gaaaaaaggc
     1021 tttgcccccg acgataaatc ccacgcggct ttacaagcga tcgggggcac cctctctctg
     1081 gctgaacgag cagtttggaa agaagttcag gctcagttac gctggaaatg gcatacattt
     1141 tttaaaagct atgatgtggt gctttcaccg gttctcatgc ggcctgcttt tgagcacaac
     1201 catcagtcga attggcacaa aagagaactg gatgtgaatg gtgttaaacg tccttacatg
     1261 gatgtgttaa tttgggctgg tccagcggtt gtctcctacc tgccggctac agcggcgccg
     1321 gtcggcgtga caagtgaagg gaagcctgtg ggtattcaga ttattggccc acacttagaa
     1381 gactatacca caattgctgt ggcggggatg ttcgaagaaa tcctgggagg attcaaaccg
     1441 ccgaagggtt gggctgcagc gctcgaactc gagcaccacc accaccacca ctgagatccg
     1501 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1561 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     1621 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1681 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1741 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1801 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1861 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1921 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1981 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2041 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     2101 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2161 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     2221 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     2281 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     2341 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     2401 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     2461 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     2521 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     2581 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     2641 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     2701 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     2761 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     2821 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     2881 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     2941 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     3001 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     3061 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3121 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3181 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3241 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3301 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3361 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3421 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3481 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3541 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3601 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3661 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3721 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3781 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3841 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3901 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     3961 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     4021 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4081 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     4141 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     4201 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     4261 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     4321 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     4381 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     4441 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     4501 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     4561 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     4621 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     4681 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     4741 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     4801 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     4861 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     4921 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     4981 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     5041 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     5101 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5161 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5221 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     5281 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     5341 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     5401 cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc
     5461 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     5521 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     5581 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     5641 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     5701 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     5761 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     5821 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     5881 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     5941 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     6001 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     6061 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     6121 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     6181 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     6241 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     6301 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     6361 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     6421 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     6481 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     6541 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatcgatctc
     6601 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     6661 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatg
//
