LOCUS       sequence-426043-        6698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for MsmegA gene, codon optimized for expression
            in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6698)
  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 6698)
  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: MsmegA _pET29b
FEATURES             Location/Qualifiers
     source          1..6698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1468..1485
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1538..1556)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1552..1599
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1636..2091
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1723..1742)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1933..1954
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2183..2998)
                     /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(2183..2998)
                     /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     2906..2925
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2990..3009
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3120..3708
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3609..3628
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3862..3879
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3894..4033
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3959..3978)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4119..4141
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4135..4326)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5135..6217)
                     /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     6179..6198
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6218..6295)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6501..6520
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6608..6627
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6608..6626
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6627..6651
                     /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             6666..6688
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 aaattatcgg aatattgcag tcatgatgcc tcaggtctgg ccgaactggt gcgtacggga
       61 gaagtgactg cattagagct ggccaagctg gcgttagaag gcattaatat tctgaatcca
      121 gatctgaacg ccgttctgga agtgttcgat gatcagctgg agaaactggc ggaaaccggc
      181 actaaagatg ctccattctt aggagtacca tttctggtta aagatcttat attgcatata
      241 ggtggacgga aaaatgaaat gggttcacgt ctgtgtcaag gcctcgtgtc tccgaacaac
      301 actgacctgg caactaagta cgacgaagcg ggactcatca cactcggtag aaccgccacc
      361 ccggaactgg gtttttgtac gacgactgaa tcgttgcttt cagggccaac ccgaaatcca
      421 tggaatatta atatgatgcc aggaggttct agcgggggta gcgcggcgat ggtggcagcc
      481 ggtgccgtac cagtagccca tgggaatgat ggcggagggt cgattcgcat tccggccgcc
      541 tgctgtggtt tagttggtct gaaaccgagt cgcgggagaa ctccaatcgg accggaagtg
      601 gccgatggct taaacggact ggggatcgag catgtcttaa cccgctccct tcgcgattgt
      661 gctgctattt tagatgcaac agagggtccc atgccaggcg atccgtatgc tattatacga
      721 cccgaaagat catatcttag tgagctggat cgtaaatgtg aacctcttaa aataggcttc
      781 acctctatgg catggagcgg agatcatgtg gatgcagaga ttactgaaaa aacgaaaaaa
      841 attgcactct tgtgcgcaga catgggccat aatgtggaag atgcatcgcc aacctttgac
      901 tatggttcat ttcgtgaagc aacggtcact ttttggtgcg cgaacatggc actttggatt
      961 acccaaataa gtgagatgac tggtcggcct gcgaaccgcg aaacactcga ggccaccact
     1021 cttgcatgtt accagtatgg gctgggcttg aaagcgacag atatggccac cgcatttgct
     1081 gtgatgaaca tgatcagtag agatgtttcc gtctttttcc agaaatatga cgttttgatt
     1141 accccaacca cttcactgct tccgcagaag actggcactt atgacgcgaa cgactctagc
     1201 ttcgatgctc acggctggac tgatcatatt tgctctttcg ctccttttac ggcattgttt
     1261 aatatgactg gccagccggc catttcactt cctctttcta tgtctgcaac aggtctgccc
     1321 atcgggaccc agtttgtcgc gggtagcggt cgcgaagata tcctgtttcg cttgtcaagt
     1381 cagctcgaac aggcgttgcc ctgggcagat agacatccta aaacgtccct gtggaatgac
     1441 gcgtacatgg gcaaaagcgt actcgagcac caccaccacc accactgaga tccggctgct
     1501 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1561 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     1621 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     1681 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     1741 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     1801 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     1861 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     1921 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     1981 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2041 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     2101 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2161 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     2221 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     2281 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     2341 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     2401 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     2461 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     2521 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     2581 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     2641 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     2701 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     2761 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     2821 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     2881 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     2941 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     3001 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     3061 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3121 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3181 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3241 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     3301 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3361 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3421 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3481 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3541 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3601 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3661 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3721 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3781 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3841 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     3901 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     3961 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4021 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4081 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4141 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     4201 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     4261 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     4321 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     4381 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     4441 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     4501 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     4561 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     4621 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     4681 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     4741 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     4801 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     4861 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     4921 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     4981 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     5041 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     5101 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5161 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5221 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     5281 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     5341 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     5401 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     5461 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     5521 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     5581 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     5641 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     5701 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     5761 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     5821 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     5881 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     5941 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     6001 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     6061 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     6121 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     6181 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     6241 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     6301 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     6361 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     6421 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     6481 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     6541 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc
     6601 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
     6661 ataattttgt ttaactttaa gaaggagata tacatatg
//
