LOCUS       sequence-426041-        6548 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct for UMG-SP-1 gene, codon optimized for 
            expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6548)
  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 6548)
  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-1_pET29b
FEATURES             Location/Qualifiers
     source          1..6548
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1318..1335
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1388..1406)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1402..1449
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1486..1941
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1573..1592)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1783..1804
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2033..2848)
                     /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(2033..2848)
                     /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     2756..2775
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2840..2859
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2970..3558
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3459..3478
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3712..3729
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3744..3883
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3809..3828)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3969..3991
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3985..4176)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4985..6067)
                     /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     6029..6048
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6068..6145)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6351..6370
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6458..6477
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6458..6476
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6477..6501
                     /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             6516..6538
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gctagagaac caaccgcgtt ggccacggcc gcggcaattc gtagtggcgc gaccaccgct
       61 cgagctgaga cggaggctgc cattgcccgt attgaagctc tcgatggcgc cattaacgcc
      121 gtagtagtgc gagacttcga tcgagctctc gcggcagcag atgccgccga cgcgcgaatt
      181 caagctggag atacagcacc attattaggt gttccgatga ctgtcaaaga ggcatttgac
      241 gttgaaggcc tgccgacgca ttggggattc cgccagcacg ccgggaatat agcaacaagc
      301 gacgcggaag cagtgagacg cctgaaagca gcgggagcga ttatccttgg taagacgaac
      361 gtaccaaaag gcctgggcga ttggcaaagt gtaaactcga ttcatggcgt tacgaatcac
      421 ccattagatc ccacccgtac accaggaggc tcatcagggg gttctgcagc agccttggca
      481 agcggaatgg tcccgctgga gctgggatca gatattggcg ggtcaataag aattccagcg
      541 cacttctgtg gagtgtgggg cctcaaacca tcctggggcg caatatcctc gcacggtcat
      601 cgttatccag gaacaaatgg cgcagaaact cccttagggg taatcgggcc gatggcccgc
      661 agtccggacg atttggcggc catgctggat ttgctggcta cgctgccgat gcctcgtgct
      721 tcccgacctc ctcgtagagt tttagctatt acagaccatc cagctattcg cacctccgcc
      781 gtgtgccgtg atgccgtgga caccgcggct gaagctctgg caggtgcggg aattgaagtg
      841 atacgaagta cggatctgtt gccggatctc gctcgtcaac atcatgcata tgggcagatg
      901 ctgtcagttg catttgcacg cagcgaccca acactgcatg ccagtctccc taaccttttg
      961 acctggttga gttggcagga tgcacaagct cggaatacac gtgcgtgggg ccgtctgttt
     1021 ggggaagttg atgcagtgat cgcaccgccg gcagccaccc aagcttttgc acatgaccac
     1081 gcaccacaag ctaatagaac tttagatatc gatggtgtgg cgtctccata cgacgctcat
     1141 ctggcatggg ctggagtggc tacctatccg ggtctgcccg ctgtggtggt tccggtgggc
     1201 acagccaatg gattgccagt tggtgtccaa gtaattacgg attttcaccg cgatcacgac
     1261 gctattgcaa ccgctgcctt aattcatcgt ctgactgaag gtcagccggc actcgagcac
     1321 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     1381 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1441 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     1501 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     1561 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     1621 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     1681 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1741 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1801 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1861 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     1921 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1981 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     2041 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     2101 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     2161 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     2221 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     2281 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     2341 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     2401 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     2461 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     2521 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     2581 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     2641 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     2701 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     2761 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     2821 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     2881 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2941 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3001 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3061 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     3121 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3181 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3241 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3301 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3361 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3421 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3481 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3541 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3601 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3661 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3721 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3781 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3841 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     3901 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3961 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4021 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     4081 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     4141 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     4201 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     4261 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     4321 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     4381 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     4441 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     4501 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     4561 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     4621 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     4681 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     4741 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     4801 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     4861 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     4921 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     4981 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5041 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5101 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5161 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5221 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
     5281 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5341 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5401 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5461 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5521 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5581 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5641 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5701 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5761 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5821 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     5881 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     5941 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     6001 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     6061 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     6121 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6181 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6241 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6301 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     6361 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     6421 gcgtagagga tcgagatcga tctcgatccc gcgaaattaa tacgactcac tataggggaa
     6481 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata
     6541 tacatatg
//
