LOCUS       sequence-426250-        6302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for AfNitB gene, codon optimized for expression
            in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6302)
  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 6302)
  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: AfNitB_pET29b
FEATURES             Location/Qualifiers
     source          1..6302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1072..1089
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1142..1160)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1156..1203
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1240..1695
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1327..1346)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1537..1558
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1787..2602)
                     /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(1787..2602)
                     /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     2510..2529
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2594..2613
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2724..3312
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3213..3232
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3466..3483
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3498..3637
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3563..3582)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3723..3745
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3739..3930)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4739..5821)
                     /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     5783..5802
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5822..5899)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6105..6124
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6212..6231
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6212..6230
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6231..6255
                     /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             6270..6292
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 cagaccagaa aaattgttcg ggccgcagcc gtccaggccg cgtctcctaa ttatgacctg
       61 gcagctggtg tagataaaac aatcgaatta gctagacagg ctcgtgacga agggtgtgac
      121 ttaatcgtct ttggcgaaac atggctgccc ggttatccct ttcatgtctg gttaggtgcc
      181 cctgcctggt cattgaaata tagtgcccgc tattatgcga attctttgtc gctggactca
      241 gtggagtttc aaagaattgc gcaagccgcg cgcaccttag gcatatttat tgctttaggt
      301 tatagtgagc gcagtggggg ctccctctat ctgggccaat gcctgattga tgataaaggc
      361 gaaatgctgt ggtcccggcg taaactgaaa ccaacccacg tcgaacgcac cgttttcgga
      421 gaaggctatg cgcgtgatct gattgtatct gaaactgagc ttggacgtgt tggggccctg
      481 tgctgctggg agcatctgtc tccattaagc aaatacgctc tgtattccca gcatgaagcc
      541 attcacatcg ctgcctggcc ttcattttca ttgtattcag aacaggcaca tgctttatcc
      601 gcgaaagtga acatggccgc cagccagatc tattctgtag agggtcagtg ttttaccatc
      661 gcggcaagtt ccgtggtaac gcaagaaact ctcgatatgc ttgaagtggg ggaacacaat
      721 gcctctctgc tgacggtggg gggtggttct agtatgatct ttgcaccgga cggcagaacc
      781 ctcgccccgt atctgcccca cgacgcggaa ggtttaatca tcgctgatct gaacatggag
      841 gaaatcgcct tcgcgaaggc cataaatgat cccgcaggcc attactcaaa accggaagcc
      901 acccgtctcg tgttagatct tggacatcgt gagcccatga cgcgtgtcca ctcacaaagc
      961 ccgatcaaag aagaggcctg cgaacctctg acgccctcta ccattgctcc ggtagcgatt
     1021 agtcagatcc aggaaagtga cacgttgttg gtgcaggagc cctccctcga gcaccaccac
     1081 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     1141 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1201 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     1261 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1321 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1381 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1441 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1501 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1561 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1621 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1681 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1741 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     1801 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     1861 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     1921 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     1981 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     2041 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     2101 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     2161 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     2221 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     2281 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     2341 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     2401 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     2461 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     2521 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     2581 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     2641 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2701 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2761 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2821 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     2881 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2941 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3001 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3061 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3121 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3181 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3241 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3301 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3361 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3421 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3481 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3541 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     3601 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     3661 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3721 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     3781 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     3841 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     3901 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     3961 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     4021 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     4081 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     4141 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     4201 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     4261 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     4321 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     4381 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     4441 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     4501 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     4561 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     4621 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     4681 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     4741 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4801 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     4861 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     4921 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     4981 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
     5041 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     5101 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     5161 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     5221 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     5281 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     5341 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     5401 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     5461 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     5521 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     5581 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     5641 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     5701 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     5761 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     5821 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     5881 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     5941 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     6001 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     6061 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     6121 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     6181 aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     6241 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata
     6301 tg
//
