LOCUS       sequence-425823-        6539 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for NylB' gene, codon optimized for expression 
            in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6539)
  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 6539)
  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: NylB'_pET21b
FEATURES             Location/Qualifiers
     source          1..6539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1180..1197
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1250..1268)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1264..1311
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1348..1803
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1435..1454)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1645..1666
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1829..1933
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1934..2002,2003..2794)
                     /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(2152..2171)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2965..3553
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3454..3473
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3707..3724
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3739..3878
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3804..3823)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3964..3986
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3980..4171)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4980..6062)
                     /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     6024..6043
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6063..6140)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6346..6365
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6449..6468
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6449..6467
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6468..6492
                     /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             6507..6529
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 aacgcgcgtt caaccggtca gcatcctgcc cgctatccag gcgcggccgc aggcgagccc
       61 accctcgata gttggcagga accaccacat aaccgttggg ctttcgcgca tttgggtgaa
      121 atggtcccat ctgctgccgt ctcgagacgt ccggttaacg ctccgggaca cgcgttggcc
      181 cgtttgggag caattgccgc ccagctgccg gacttggaac aacgtttgga acagacgtat
      241 acagacgcat ttctggtatt acggggcacc gaagtggtgg cggaatatta tagagcaggg
      301 tttgcccctg acgaccgcca ccttctgatg agcgtgtcga aatccctctg tggcaccgtg
      361 gtgggcgcac tcgtcgatga aggccgtatt gatccggcac agccagtcac ggaatacgtg
      421 cccgagctgg cgggtagcgt ctatgatgga ccctctgttt tacaggtctt ggatatgcag
      481 atttcaattg attacaacga agactatgtt gacccggcgt cagaggttca aactcacggc
      541 cggtctgcag ggtggcgtac ccgccgtcat ggggatccgg ccgacacgta tgaattcctg
      601 accacacttc gtggtgatgg ctccaccggt gagtttcaat attgctccgc caatacagat
      661 gtgctggcct ggattgtgga gcgtgttacc gggcttcgtt atgtagaagc cctgtcaaca
      721 tatttgtggg ccaaactgga tgcggatcgt gatgcgacta tcacagttga taccaccggc
      781 ttcggtttcg ctcatggcgg tgtgtcgtgc acagcacggg atctggcccg tgtcggtcgt
      841 atgatgttag acggtggggt ggcacccggg ggtcgcgttg tttcagaaga ctgggtccgt
      901 cgtgttctgg ctggcggatc acatgaagca atgacggaca agggctttac aaatacgttt
      961 cccgatggct catatacgcg gcaatggtgg tgtacaggca atgagcgggg taatgtttcc
     1021 ggaataggta tccacggaca gaatttatgg ctcgatccat taacagacag cgtcatagtc
     1081 aagttgtctt cttggccgga cccagatact gagcactggc accgcctgca gaatggcatc
     1141 ctgctggatg tcagccgagc cctggatgct gttctcgagc accaccacca ccaccactga
     1201 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     1261 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     1321 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     1381 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1441 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1501 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1561 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1621 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1681 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1741 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     1801 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1861 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1921 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     1981 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     2041 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     2101 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     2161 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     2221 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     2281 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     2341 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     2401 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     2461 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     2521 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     2581 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     2641 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     2701 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     2761 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     2821 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     2881 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2941 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3001 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3061 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3121 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3181 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3241 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3301 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3361 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3421 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3481 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3541 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3601 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3661 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3721 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3781 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     3841 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     3901 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3961 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4021 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     4081 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     4141 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     4201 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     4261 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     4321 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     4381 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     4441 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     4501 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     4561 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     4621 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     4681 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     4741 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     4801 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     4861 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     4921 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     4981 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5041 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     5101 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     5161 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     5221 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     5281 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     5341 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     5401 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     5461 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     5521 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     5581 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     5641 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     5701 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     5761 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     5821 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     5881 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     5941 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     6001 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     6061 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     6121 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     6181 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     6241 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     6301 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     6361 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     6421 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     6481 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatg
//
