LOCUS       sequence-426047-        6551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for UMG-SP-2 gene, codon optimized for 
            expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6551)
  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 6551)
  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: UMG-SP-2_pET29b
FEATURES             Location/Qualifiers
     source          1..6551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(203..214)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             1321..1338
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1391..1409)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1405..1452
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1489..1944
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1576..1595)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1786..1807
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2036..2851)
                     /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(2036..2851)
                     /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     2759..2778
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2843..2862
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2973..3561
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3462..3481
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3715..3732
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3747..3886
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3812..3831)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3972..3994
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3988..4179)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4988..6070)
                     /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     6032..6051
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6071..6148)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6354..6373
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6461..6480
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6461..6479
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6480..6504
                     /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             6519..6541
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tctgaactca gtgctatcga aacggctgct gcgattgccg gagggtcaat gacggcgctg
       61 gaggcgtgcg atgcggcaat agcccgcata gagcagcgcg atggcccaat aaacgccgtg
      121 gtggttcgcg atttcgatcg cgcacgtgat gccgctaaag cggccgatgc agaaatcgct
      181 gctgccgtac gcaaacctct gcttggagtt cccatgacca taaaagaatc ctttgacatc
      241 gcaggtctgc cgactagctg gggctttgcc gaacatgcag atcatattgc cactgcagat
      301 tccctggtag taagtcgtct gaaggccgca ggcgcggtat ttctgggaaa atcaaatatt
      361 ccagtgggtt tagctgactg gcagagcgtg aatcccaact atgggcgcac caacaatccg
      421 catgatcaca gccgttccgc aggtggaagc tcaggtggag cagccgcggc cctggccgcc
      481 ggcatggtac ccttagaata cggctctgat attggcggta gcattcgcgt tccggcacac
      541 ttctgtggtg tatggggcct caaaaccact tttgacgctg tttctttaga agggcattat
      601 tttccgagaa ccgattccgc gaaagctgat cttagcgtag tcggccccat ggctcgcacc
      661 cctgcggatt tggcgttagc ccttgacata acgtctaaag tgccattacc tcaaagtcgt
      721 atagctaatc ttagtggact gcgaatattg ctgttaactg cgcatccgga aacagttgcg
      781 gattccgcga cgatttcagc ggttgagcga gccgcagcgg cctgtgaagc tagcggtgcg
      841 actgttgcta ctagctcccc tgatctgcca gacttgagtg cgttggtggc cgattacact
      901 agaatgctgc tggtagtgtt agcacggggt ctggcgcccg agggtaccga acctgttagc
      961 ctgaatgctt ggtatgccat gcttgatgac caggcccgta tgatgcgagc gtttgatcgg
     1021 ctctttgaaa gcttcgatgc tatcttctgt ccagtcctgg gaacaacggc tttcgcccat
     1081 tcggacgagc cagactgggc caagcgctcg ctcagcatcg atggaggtat cgcgccgttt
     1141 gccgcacagc tgggttggat ctccatggcg acgtacggcg gaatgccagc gctgtcaatg
     1201 cctctgggtg cagatgggaa cgggctgccg attaacctcc aaataatcac gcgcaactgg
     1261 tcagaccacg acgcaattcg cattggtgcg ctggttgcgg aggcgctgga cagactcgag
     1321 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     1381 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1441 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     1501 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1561 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1621 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1681 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1741 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1801 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1861 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1921 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1981 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     2041 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     2101 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     2161 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     2221 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     2281 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     2341 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     2401 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     2461 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     2521 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     2581 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     2641 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     2701 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     2761 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     2821 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     2881 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2941 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3001 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3061 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     3121 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3181 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3241 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3301 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3361 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3421 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3481 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3541 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3601 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3661 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3721 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3781 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3841 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     3901 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3961 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4021 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     4081 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     4141 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     4201 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     4261 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     4321 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     4381 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     4441 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     4501 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     4561 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     4621 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     4681 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     4741 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     4801 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     4861 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     4921 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     4981 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5041 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     5101 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     5161 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     5221 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa
     5281 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     5341 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     5401 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     5461 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     5521 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     5581 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     5641 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     5701 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     5761 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     5821 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     5881 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     5941 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     6001 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     6061 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     6121 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     6181 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     6241 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     6301 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     6361 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     6421 ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat taatacgact cactataggg
     6481 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag
     6541 atatacatat g
//
