LOCUS       sequence-426039-        6650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for ReU gene, codon optimized for expression in
            E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6650)
  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 6650)
  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: ReU_pET29b
FEATURES             Location/Qualifiers
     source          1..6650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1420..1437
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1490..1508)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1504..1551
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1588..2043
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1675..1694)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1885..1906
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2135..2950)
                     /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(2135..2950)
                     /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     2858..2877
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2942..2961
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3072..3660
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3561..3580
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3814..3831
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3846..3985
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3911..3930)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4071..4093
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4087..4278)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5087..6169)
                     /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     6131..6150
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6170..6247)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6453..6472
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6560..6579
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6560..6578
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6579..6603
                     /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             6618..6640
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 aatactagtg gccttggctg gatgtcagcc accgagatgg cggcccaagt tgccagtaaa
       61 aaactgagtc ctaacgagat cgcagaagaa atgatccgcc gcgttgggga ggtcaacccc
      121 gcagtcaatg caattgtaca cttcgacgcc gatcaagtta gacgcgatgc aggtgagtta
      181 acccgtgcgc aggattccgg agaaccgctg gggccattac acggtgtccc attcactatt
      241 aaagatctca cagatgtgcg gggtctgcca actaccttcg gtttgaaacc tatgagagac
      301 aacatagccg agcgagatgc tgttattgtt acccgcctga gacaagctgg gggattatac
      361 ttgggtaaaa ctaatacccc agaatcaggc tattatggtg gaacggacaa tcacttattt
      421 ggtccaacgc acaatccgtg gaaaccgggg catagcgctg gtgggtcaag tggcggagct
      481 gcggctgccg ttgcggctgg tctgggtcct ttagccgaag ggtctgatgg agctggttca
      541 gttcgcattc ctagtgccct ctgtggggtc gtaggactta agccgaccac gggtgttata
      601 cctcaaacta tactgcccgg gcggtataat aattgggcat atcacggtcc gattacccgg
      661 acagttgcag ataacgcact tatgctcgat gtactggccg gaccagatca ctcagatccc
      721 ctgtcaatag aaagagtaga aagcagctac gtggaagcgg cccgcggagg gatagatggc
      781 ttgagagttg catggagccc taatcttggc ctggggcacg tagaaccgga tgtcgcagcg
      841 gtctgtgccg aagcggtggc gtgcttcgaa gatatgggcg ccaaggtggt tgaagcaaca
      901 ccagattggg gtgatccgtc tgaagctatg tggcatggta tctgggttcc aggattcgcc
      961 ggtgaacatg atatgctcga ttgggattct ttgcatggcc aggttgatga taatctgatc
     1021 gaactgattc atgaagggcg ccggctgacc ggtgtggatt acggccgagc cgatgccttc
     1081 cgtggccgta tgtgggatac ttggacagag tttatgaatg actacgacgt acttatctca
     1141 ccaacgcttg cgagcgcgac ctttcctctg actcaatttg ctccggattg gttacaaggt
     1201 aagagtctgc gcgaacagtt gctggattgg cttctcacgt acccctacaa catgctgaac
     1261 aatcctgcta taaccgttcc cgcgggtttt acggctgatg gacgcccagt cggactgcag
     1321 atcgctgctc gccaccgcca agatgcctta gttctgcgtg tcgcagcaaa tctggaacag
     1381 gcacgcccgt gggccgatcg ccggccggtt gctctcgagc accaccacca ccaccactga
     1441 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     1501 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     1561 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     1621 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1681 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1741 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1801 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1861 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1921 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1981 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     2041 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2101 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     2161 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     2221 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     2281 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     2341 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     2401 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     2461 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     2521 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     2581 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     2641 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     2701 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     2761 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     2821 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     2881 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     2941 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     3001 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3061 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3121 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3181 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3241 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3301 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3361 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3421 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3481 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3541 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3601 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3661 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3721 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3781 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3841 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     3901 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     3961 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     4021 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4081 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     4141 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     4201 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     4261 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     4321 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     4381 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     4441 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     4501 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     4561 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     4621 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     4681 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     4741 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     4801 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     4861 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     4921 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     4981 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     5041 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     5101 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5161 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     5221 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     5281 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     5341 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     5401 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     5461 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     5521 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     5581 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     5641 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     5701 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     5761 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     5821 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     5881 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     5941 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     6001 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     6061 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     6121 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     6181 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     6241 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     6301 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     6361 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     6421 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     6481 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     6541 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     6601 tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg
//
