LOCUS       sequence-433776-        6413 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 6413)
  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 6413)
  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_pET29b
FEATURES             Location/Qualifiers
     source          1..6413
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1183..1200
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1253..1271)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1267..1314
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1351..1806
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1438..1457)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1648..1669
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1898..2713)
                     /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(1898..2713)
                     /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     2621..2640
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2705..2724
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2835..3423
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3324..3343
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3577..3594
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3609..3748
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3674..3693)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3834..3856
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3850..4041)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4850..5932)
                     /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     5894..5913
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5933..6010)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6216..6235
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6323..6342
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6323..6341
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6342..6366
                     /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             6381..6403
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgaacgctc ggtcgacggg acagcatccg gcgcgttatc cgggcgcggc cgcaggagaa
       61 cctactctgg attcgtggca ggaagccccg cacaaccgct gggcctttgc gcgtctgggt
      121 gagctgcttc ccacagcggc tgtgtcaaga cgtgatccgg cgacaccagc ggaaccggtt
      181 gttcggctgg acgcgttagc gacacgcctg ccggatctgg aacagcgctt agaggaaacc
      241 tgcaccgacg catttctcgt tttaagaggg tcagaagtat tagctgaata ttatcgtgca
      301 gggtttgctc cggatgaccg tcatcttctt atgtctgtga gtaaatcctt atgcggcact
      361 gtcgtgggcg ctctgattga cgaaggcaga atagatccgg ctcagcccgt gactgagtat
      421 gtcccagaac ttgccgggag tgtctatgat ggcccgagcg tgttacaggt cctggatatg
      481 cagatatcta tcgattataa cgaggattat gtggatcctg ctagcgaagt gcagacacat
      541 gaccgttcag caggttggcg cacccgtcgc gatggagatc cagccgatac atatgaattc
      601 ctgactaccc tgcgtggcga tggtggtacg ggagagtttc aatattgttc ggcgaacaca
      661 gatgttctcg cgtggatagt cgaacgtgta actggactgc gctatgttga ggcgcttagc
      721 acatatctct gggctaagtt ggacgcggat cgcgatgcta ctattacggt ggatcaaacg
      781 ggttttggat ttgcaaatgg tggcgtgagt tgtacagcgc gggatctggc acgggtaggc
      841 cggatgatgc tggatggagg ggttgccccg gggggccgtg tagtaagtca ggggtgggtg
      901 gaatccgtcc ttgcgggggg ttcgcgggag gctatgactg acgaaggctt tacctcagcg
      961 tttcccgaag gtagctacac gcgacagtgg tggtgtactg ggaacgaacg gggtaacgta
     1021 tctggaattg gtatccatgg acagaactta tggctggatc ctcgtaccga ctccgtgata
     1081 gttaaacttt catcatggcc agatcccgat acacgccatt ggcacggctt gcaatcaggg
     1141 attttactgg acgtttcccg tgctcttgat gctgtactcg agcaccacca ccaccaccac
     1201 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     1261 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     1321 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     1381 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     1441 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     1501 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     1561 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     1621 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     1681 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     1741 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     1801 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1861 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     1921 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     1981 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     2041 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     2101 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     2161 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     2221 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     2281 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     2341 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     2401 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     2461 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     2521 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     2581 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     2641 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     2701 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     2761 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2821 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2881 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2941 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3001 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3061 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3121 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3181 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3241 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3301 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3361 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3421 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3481 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3541 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3601 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     3661 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     3721 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     3781 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3841 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     3901 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     3961 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     4021 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     4081 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     4141 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     4201 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     4261 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     4321 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     4381 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     4441 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     4501 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     4561 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     4621 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     4681 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     4741 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     4801 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     4861 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4921 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     4981 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     5041 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     5101 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg
     5161 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     5221 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     5281 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     5341 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     5401 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     5461 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     5521 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     5581 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     5641 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     5701 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     5761 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     5821 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     5881 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     5941 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     6001 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     6061 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     6121 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     6181 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     6241 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     6301 atcgatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     6361 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat atg
//
