LOCUS       sequence-426036-        6335 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for RoL10X gene, codon optimized for expression
            in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6335)
  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 6335)
  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: RoL10X_pET29b
FEATURES             Location/Qualifiers
     source          1..6335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1045..1053
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             1105..1122
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1175..1193)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1189..1236
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1273..1728
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1360..1379)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1570..1591
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1820..2635)
                     /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(1820..2635)
                     /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     2543..2562
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2627..2646
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2757..3345
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3246..3265
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3499..3516
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3531..3670
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3596..3615)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3756..3778
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3772..3963)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4772..5854)
                     /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     5816..5835
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5855..5932)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6138..6157
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6245..6264
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6245..6263
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6264..6288
                     /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             6303..6325
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gtgccggtaa gtggcaagtc tggcagcagc acaacggctg taagcgcaag tgataactcg
       61 gcgttacccc cgctgataag cagcagatgc gcgccgccgt caaataaagg ttcgaagtct
      121 gatcttcaag cagagccgta ttatatgcag aaaaatactg aatggtacga atctcacggt
      181 ggtaacttaa catctatcgg gaaacgtgac gataacttag taggtggtat gaccctggat
      241 cttccgtcag atgccccacc catcagtttg agtggaagca cgaatagcgc ctcagatggc
      301 ggtaaggttg ctgccgccac cacggcgcag atacaagagt ttacaaaata cgcgggcatc
      361 gctgccaccg cgtattgccg gtcagtcgtt ccgggcaaca aatgggattg tgtgcagtgt
      421 cagaagtggg tgcctgacgg gaagatcatt acaacgttca cgagtctgct gtcggatact
      481 aacggttacg tcttgcgctc ggataaacag aagaccattt atttagtctt tagaggcacg
      541 aatagctttc gtagcgccat aacggacatt gtttttaatt tcagcgacta taaacccgtc
      601 aagggtgcaa aagttcacgc aggcttctta tcctcttatg agcaagttgt taatgactat
      661 tttccggtaa tacaggaaca gctcacggcg aacccgactt acaaggtcat cgtaacgggt
      721 cactctcttg ggggggcaca ggctctgctg gctgggatgg atctgtatca gcgcgagccg
      781 cgcctgtcgc caaaaaatct gagcatttac actttcggat gcccgcgcgt tggaaatccg
      841 acctttgcgt attatgtatg ttctactggt ataccattcc atagaaccgt gcacaaacgt
      901 gatattgtgc ctcatctgcc acctcaatct tttgggtacc tgcacccggg cgttgaatcg
      961 tggataaagt ctggcacgtc aaatgtacag atttgcacgt cctgtataga gacaaaagat
     1021 tgctccaatt caattgttcc ctttacctcc ttactggatc atctgagtta ctttggtatc
     1081 aacgaaggcc tgtgcctcct cgagcaccac caccaccacc actgagatcc ggctgctaac
     1141 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     1201 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     1261 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1321 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1381 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1441 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1501 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1561 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1621 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1681 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     1741 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     1801 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     1861 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     1921 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     1981 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     2041 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     2101 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     2161 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     2221 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     2281 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     2341 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     2401 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     2461 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     2521 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     2581 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     2641 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     2701 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2761 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2821 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2881 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     2941 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3001 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3061 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3121 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3181 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3241 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3301 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3361 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3421 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3481 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3541 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     3601 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     3661 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3721 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3781 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     3841 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     3901 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     3961 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     4021 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     4081 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     4141 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     4201 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     4261 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     4321 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     4381 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     4441 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     4501 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     4561 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     4621 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     4681 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     4741 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4801 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4861 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     4921 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     4981 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     5041 ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     5101 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     5161 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     5221 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     5281 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     5341 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     5401 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     5461 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     5521 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     5581 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     5641 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     5701 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     5761 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     5821 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     5881 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     5941 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     6001 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     6061 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     6121 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     6181 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg
     6241 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     6301 attttgttta actttaagaa ggagatatac atatg
//
