LOCUS       sequence-479222-        8839 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  Expression an ELP-tagged fusion enzyme of GalK and AtUSP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8839)
  AUTHORS   Wu X, Liu J, Yin X, Ma D, Zhang S, Liu X
  TITLE     Protein Fusion of Biosynthetic Enzymes and a Thermo-Responsive 
            Polypeptide Expedites Facile Access to Biocatalysts for Nucleotide 
            Sugars.
  JOURNAL   Chembiochem. 2025 Jan 13:e202401005. doi: 10.1002/cbic.202401005.
  PUBMED    39805738
REFERENCE   2  (bases 1 to 8839)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 22, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Chembiochem. 2025 Jan 13:e202401005. doi: 10.1002/cbic.202401005."
COMMENT     Sequence Label: pET30a-GalK-USP-ET64
FEATURES             Location/Qualifiers
     source          1..8839
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(559..1374)
                     /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(559..1374)
                     /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     1282..1301
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1366..1385
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1496..2084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1985..2004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2238..2255
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2270..2409
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2335..2354)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2495..2517
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2511..2702)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3511..4593)
                     /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     4555..4574
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4594..4671)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4877..4896
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4984..5003
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4984..5002
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5003..5027
                     /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             5041..5063
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             8683..8700
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(8753..8771)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8767..8814
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      541 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
      601 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
      661 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
      721 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
      781 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
      841 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
      901 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
      961 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1021 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1081 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1141 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1201 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1261 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     1321 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     1381 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     1441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1621 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     1921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     1981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2281 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     2341 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     2401 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2461 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     2521 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     2581 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     2641 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     2701 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     2761 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     2821 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     2881 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     2941 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3001 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3061 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     3121 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     3181 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     3241 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     3301 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     3361 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     3421 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     3481 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3541 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3601 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     3661 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     3721 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     3781 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     3841 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     3901 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     3961 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4021 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4081 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4141 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4201 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4261 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     4321 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4381 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     4441 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     4501 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     4561 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     4621 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     4681 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     4741 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     4801 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     4861 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     4921 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatcgatctc gatcccgcga
     4981 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaataat
     5041 tttgtttaac tttaagaagg agatatacca tgggtgcaca acatcttact actgaagccc
     5101 ttcgcaaaga ctttcttgct gtttttggtc aagaagcaga tcaaaccttc ttttcaccag
     5161 gccgcattaa tttgattggt gaacacacag actacaacgg tgggcacgtt tttcctgctg
     5221 ctatttcctt gggaacttac ggtgcagctc gtaagcgtga cgaccaagtc ttgcgtttct
     5281 actcagctaa ctttgaggac aagggcatta tcgaagtgcc tctcgctgac ctcaagtttg
     5341 aaaaagagca caactggacc aattatccaa aaggtgtcct tcatttcttg caagaagctg
     5401 ggcacgtgat tgacaaaggt tttgattttt atgtttatgg aaatattcca aatggtgctg
     5461 gcttgtcttc ttctgcatcc ttggaactct tgacaggagt cgtggctgag catctctttg
     5521 atttaaaatt agagcgtctc gatttggtta aaatcggcaa acaaacagaa aacaacttta
     5581 tcggagtaaa ctctggcatt atggaccagt ttgctattgg tatgggggca gaccaacgtg
     5641 ctatttacct agatactaat actttagaat acgacttggt gccacttgat ttgaaggaca
     5701 atgtcgttgt tatcatgaac accaacaaac gccgtgaatt ggcggactct aaatacaatg
     5761 aacgtcgtgc tgagtgtgaa aaagcagtgg aagaattgca agtttccttg gatattcaga
     5821 ctctgggtga attggacgag tgggccgttg accaatatag ctatctgatt aaagatgaaa
     5881 atcgtttgaa acgtgctcgc catgctgtgc ttgaaaacca acgtaccctc aaagctcaag
     5941 tagcactcca agcaggagat ttggaaacat ttggacgctt gatgaatgcg tcacacgttt
     6001 ctctggagca tgattatgaa gtaactggtt tggaattgga tacccttgtt cacacagctt
     6061 gggcacaaga aggagttctc ggtgctcgta tgacaggggc tggttttggt ggctgtgcca
     6121 ttgccttggt tcaaaaagat actgttgagg cctttaagga agctgtaggc aaacactacg
     6181 aggaagtagt tggatacgct ccaagcttct atatcgctga agttgcaggt ggcactcgcg
     6241 tccttgacgg tagcagcggc ggatccggtg cttctacggt tgattccaat ttcttctcat
     6301 ccgtcccagc tcttcacagt aacctcggac ttttgtctcc tgatcagatc gaactggcga
     6361 agatcttatt agaaaatggt cagagtcatc tcttccaaca atggcctgaa ctcggtgttg
     6421 atgataaaga gaagctagct tttttcgatc agattgctcg cttaaattca agctatcctg
     6481 gaggtttagc agcatacatt aagactgcga aagagcttct cgctgattcg aaagtaggga
     6541 aaaacccata tgatggtttc tctccttctg tcccttcagg agaaaatctc acttttggta
     6601 ctgacaactt catcgaaatg gaaaaaagag gtgtcgtcga agctaggaat gctgcttttg
     6661 ttctggttgc tggtggtctt ggcgagcgtc ttggttacaa tggaatcaag gtggcacttc
     6721 caagggagac aactacaggg acatgcttct tgcagcatta cattgaatct atattggctc
     6781 ttcaagaagc tagcaacaag atcgattctg atggaagtga gagagacatt cctttcatta
     6841 ttatgacatc tgatgatact cattcacgta cactggacct gttagagtta aactcttatt
     6901 ttggaatgaa acctactcaa gtacatcttc taaaacagga aaaagttgca tgtcttgatg
     6961 ataacgatgc cagacttgca ttagaccctc acaacaaata cagtatccag acaaaacctc
     7021 atggacatgg tgatgtacat tcgcttcttt attccagtgg ccttcttcat aaatggcttg
     7081 aggctggttt aaaatgggtt ttgtttttcc aagatacaaa tggacttctc ttcaatgcaa
     7141 ttccagcttc tttgggtgtg agtgctacca aacagtatca tgttaactct ctcgctgttc
     7201 ctcgcaaggc gaaggaagct attggaggaa ttagtaaact tactcacgtt gatgggagat
     7261 ctatggtaat caatgtggag tacaatcaac ttgatcctct acttagagca tctggattcc
     7321 ctgatgggga tgttaattgt gagacgggct tctccccatt cccgggaaat attaatcaat
     7381 tgattctgga acttggtccc tacaaagacg agttgcaaaa aactggaggg gccattaaag
     7441 agtttgttaa tccaaaatac aaggatagca ccaaaacagc atttaaatct tcgactcgtc
     7501 tagagtgtat gatgcaagat tatccaaaga cattgcctcc tacggcacga gttggattca
     7561 cggtgatgga tatatggctt gcttatgcac ccgtaaagaa taaccctgaa gatgctgcta
     7621 aggtaccaaa gggaaaccca tatcatagtg ctacttctgg agaaatggcg atttatcgtg
     7681 ctaacagcct cattcttcaa aaggctggtg tcaaagtgga agagcctgtg aagcaagtct
     7741 tgaatggcca agaggttgaa gtatggtctc gtatcacatg gaaacccaaa tggggaatga
     7801 tcttctctga tatcaaaaag aaagtcagcg ggaactgcga ggtctctcag agatccacaa
     7861 tggcaatcaa gggtcgtaat gtatttatca aagatctctc cttggacggt gctcttattg
     7921 tagattccat tgatgatgca gaggtaaaac ttggaggttt gataaagaac aatggttgga
     7981 ccatggagag tgtagattac aaggacacat cggttccaga agaaataaga atcagaggtt
     8041 ttagattcaa caaagtggag caactcgaaa agaagttaac tcaacctggc aaattttctg
     8101 ttgaagatgg cagcagatcc acggtctccg gtgtgggcgt tccgggcgtt ggcgtcccgg
     8161 gtgtgggcgt tccgggcgtt ggcgtcccgg gtgtgggcgt tccgggcgtt ggcgtcccgg
     8221 gtgtgggcgt tccgggcgtt ggcgtcccgg gtgtgggcgt tccgggcgtt ggcgtcccgg
     8281 gtgtgggcgt tccgggcgtt ggcgtcccgg gtgtgggcgt tccgggcgtt ggcgtcccgg
     8341 gtgtgggcgt tccgggcgtt ggcgtcccgg gtgtgggcgt tccgggcgtt ggcgtcccgg
     8401 gtgtcgggtg tgggcgttcc gggcgttggc gtcccgggtg tgggcgttcc gggcgttggc
     8461 gtcccgggtg tgggcgttcc gggcgttggc gtcccgggtg tgggcgttcc gggcgttggc
     8521 gtcccgggtg tgggcgttcc gggcgttggc gtcccgggtg tgggcgttcc gggcgttggc
     8581 gtcccgggtg tgggcgttcc gggcgttggc gtcccgggtg tgggcgttcc gggcgttggc
     8641 gtcccgggtg ttgcgcaggt gagcagcaga tcctaagtcg agcaccacca ccaccaccac
     8701 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     8761 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     8821 ggaggaacta tatccggat
//
