LOCUS       sequence-426248-        6347 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for MGS0156 gene, codon optimized for 
            expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6347)
  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 6347)
  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: MGS0156_pET29b
FEATURES             Location/Qualifiers
     source          1..6347
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             10..39
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             46..66
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1117..1134
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1187..1205)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1201..1248
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1285..1740
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1372..1391)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1582..1603
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1832..2647)
                     /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(1832..2647)
                     /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     2555..2574
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2639..2658
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2769..3357
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3258..3277
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3511..3528
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3543..3682
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3608..3627)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3768..3790
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3784..3975)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4784..5866)
                     /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     5828..5847
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5867..5944)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6150..6169
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6257..6276
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6257..6275
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6276..6300
                     /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             6315..6337
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ggcagtagcc atcaccacca ccatcaccat catcaccatt cgggagaaaa tttatatttt
       61 caaagcctgt ttcccggttg tgcgcggcgt ccagtagcgc cgctggcaca cgcaatgtcc
      121 ccatcggtcc ttgttccggc cggtctggcg ggcattcagg atgggcgcgg tcggtttcgt
      181 gaaatcatga cagctattat ggctgaccac ggcgcgttcc tgcctggcga tcgcagcagt
      241 gatggtgatg gcatcctttg gcgtttagct ggggaaccag gaccaaccgg ccgcccggtt
      301 ccactcggcg taagtacggc agggattcgt cttgttctcg tgccgggcct gcttgcggag
      361 tgcgtgtctg aatcctccct gttgttcgat gatgcacggc ctgatgttga acgctatggg
      421 tatgccacta ccctggtccg cactggcgga cgctggggta gtgcccgtaa cgcggccatt
      481 atccacgagg ttgtagcgaa acttcctgaa aatgacacga ttgtgtttgt gacccactct
      541 aaaggcgccg ttgatgtgtt ggaggccctg gtgagctatc cggatttggc tgcgcgcacg
      601 gccgccgtcg tctcggttgc gggggccatc gatggtagcc cactggctga gaccttttct
      661 gatggattgc tgcgctttgc ggaatcaatg ccgctgagta gctgtcctcc cggtgagggt
      721 actgaagcac tggattcctt gaaacgtgct taccgtctgc gcttcctggc ggaacaccgt
      781 cttccggcgc gtgtgcgcta ctatagcctc gcagcatttg caagccgcga ggaaacctcg
      841 gcaattctgc gtccatttta tgatatcttg gccaagaccg atgcgctgaa tgatggtctg
      901 gttattgccg cggatgctat tattccgggt ggcaccttgc tcggctatcc gaatgcggat
      961 catttggcag tggcaatgcc gttttctaaa aagccgtctt tgttgacctc cgtcatttcc
     1021 aagaatagtt atccgcgccc ggcactgctg gaagcaattg cccgttatgt cgaagaagac
     1081 ctgggtccgg aaaaagggcg tgaaaactaa ctcgagcacc accaccacca ccactgagat
     1141 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     1201 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     1261 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1321 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1381 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1441 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1501 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1561 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1621 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1681 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     1741 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1801 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     1861 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     1921 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     1981 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     2041 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     2101 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     2161 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     2221 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     2281 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     2341 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     2401 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     2461 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     2521 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     2581 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     2641 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     2701 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2761 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2821 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2881 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     2941 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3001 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3061 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3121 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3181 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3241 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3301 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3361 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3421 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3481 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3541 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     3601 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     3661 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     3721 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3781 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     3841 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     3901 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     3961 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     4021 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     4081 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4141 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     4201 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4261 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4321 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4381 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     4441 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     4501 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     4561 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     4621 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     4681 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     4741 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     4801 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4861 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     4921 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     4981 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     5041 tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg
     5101 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     5161 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     5221 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     5281 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     5341 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     5401 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     5461 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     5521 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     5581 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     5641 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     5701 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     5761 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     5821 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     5881 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     5941 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     6001 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     6061 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     6121 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     6181 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat
     6241 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     6301 cctctagaaa taattttgtt taactttaag aaggagatat acatatg
//
