LOCUS       sequence-425825-        6800 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for NfPolyA gene, codon optimized for 
            expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6800)
  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 6800)
  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: NfPolyA_pET21b
FEATURES             Location/Qualifiers
     source          1..6800
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1441..1458
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1511..1529)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1525..1572
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1609..2064
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1696..1715)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1906..1927
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2090..2194
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2195..2263,2264..3055)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2413..2432)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3226..3814
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3715..3734
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3968..3985
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4000..4139
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4065..4084)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4225..4247
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4241..4432)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5241..6323)
                     /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     6285..6304
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6324..6401)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6607..6626
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6710..6729
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6710..6728
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6729..6753
                     /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             6768..6790
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gatgtggcag aatacgctgc tcatgatgca acgggtctgg cagaactgat tcgtgagggt
       61 caggttagcg cggctgaagt atggacagca gctgcgacag cacttgatgc agtagagcct
      121 gaactggccg cggtcgccgg ggagcgcttc gacaaaccgc tggattatga tgagagtggg
      181 ccattcgcgg gtgtaccttt tgccctgaaa gatctgattg cacacgcagg gggagtccca
      241 agtcgttctg gctctcgcct ctttggcgcc ggcgtggctc atcctgaaga tacgcacttg
      301 gtcgcccgtt ttagacgtgc tgggctggca atcggcgcga tcactagatc tccagaattt
      361 ggattcaatg cgactacaga agccatcgcc tacgggggac cgtcacgcaa tccgtgggcg
      421 accgaccgct ctccaggcgg tagttctggc gccagcgctg cgctggttgc ctccggagcg
      481 ctgcccatgg cccatgccaa cgatgggggt ggttctattc gaattccggc tgccgcatgt
      541 ggagcggttg ggctcaagcc ttctcgtgga cgtacaactc cggggcccga tttcgcagat
      601 ccgctgttgg gtctcggcat tgagttcgca gtgacccgca cggttagaga ctgtgcgcgg
      661 ttattagatg ctgtgcatgg tgctgaaccg ggtgatcggt atttgttgcc gggcccggta
      721 agaagttatg ccgagcatgc cgccgcaggt tcccggcccc tgcggattgc tgttactact
      781 acgcctatgg acgcaggcag agccgtggac ccggagtgcg ttgctgccgt caatcgagtt
      841 gcagagcggt tagcagaatt aggccacgtt gtagaggaag cggcccctga attggatgtc
      901 gctgcctttg acaaagccaa cctggacgca tggtgctcat tcctggcgga tgcggtctta
      961 ggtgctagtg cacagttagg agtccagcct agtcgcgaat acttggaggc cactacattg
     1021 gcgtgcgtgg aatatggcaa aacgctttcg gcgtttgaca tctttacagc ggaccgtgtc
     1081 ttcaaccaga ccacacgtgc tgttgccggg tttttgaccc gttatgacgt gctcctgact
     1141 cctacaacaa gtgcaccgcc cattccactt ggacatttag acgcagacga cgcgagcctt
     1201 agtgcccgcg aatggtatga tcgtatcttc gattacggaa gctttactgc actgtttaat
     1261 gttaccggca tgcctgctat ttctctgcct cttgcagaaa gcacagcggg gctgcctatc
     1321 gggattcaat ttgccggccg atacggcgat gaggcaacat tgctggcatt ggctggagat
     1381 ttagaacgag caatgccatg ggctgatcgc cgccctgcgg tgcacgttgg ccgtctcgag
     1441 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     1501 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1561 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     1621 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1681 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1741 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1801 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1861 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1921 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1981 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2041 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2101 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2161 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2221 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2281 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     2341 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2401 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     2461 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2521 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2581 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2641 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2701 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     2761 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2821 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2881 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2941 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     3001 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     3061 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     3121 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3181 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3241 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3301 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3361 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3421 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3481 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3541 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3601 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3661 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3721 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3781 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3841 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3901 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3961 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     4021 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     4081 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4141 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4201 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4261 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     4321 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4381 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4441 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4501 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4561 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4621 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4681 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4741 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4801 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4861 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4921 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4981 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     5041 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     5101 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     5161 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     5221 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5281 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     5341 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     5401 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5461 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5521 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5581 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5641 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5701 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5761 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5821 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5881 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5941 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     6001 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     6061 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     6121 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     6181 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     6241 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     6301 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     6361 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     6421 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     6481 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     6541 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     6601 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     6661 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     6721 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     6781 aagaaggaga tatacatatg
//
