LOCUS       sequence-479229-        8746 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  Expression an ELP-tagged FKP enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8746)
  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 8746)
  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-FKP-ET64
FEATURES             Location/Qualifiers
     source          1..8746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             3153..3175
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3189..3213
                     /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)."
     primer_bind     complement(3213..3232)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3214..3232)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3320..3339)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3545..3622
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3623..4705
                     /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     complement(3642..3661)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5514..5705
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5699..5721)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5807..5946
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5862..5881
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5961..5978)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6132..6720)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6212..6231)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6831..6850)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6842..7657
                     /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             6842..7657
                     /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     complement(6915..6934)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(7749..8204)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7886..7907)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8098..8117
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(8241..8288)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     8284..8302
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(8355..8372)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 acacccggga cgccaacgcc cggaacgccc acacccggga cgccaacgcc cggaacgccc
       61 acacccggga cgccaacgcc cggaacgccc acacccggga cgccaacgcc cggaacgccc
      121 acacccggga cgccaacgcc cggaacgccc acacccggga cgccaacgcc cggaacgccc
      181 acacccggga cgccaacgcc cggaacgccc acacccggga cgccaacgcc cggaacgccc
      241 acacccggga cgccaacgcc cggaacgccc acaccggaga ccgtggatcc tgatcgtgat
      301 acttggaatc ccttatcaga taacgtcatt tcgacaaaac gtgcccgcgg attcggagcg
      361 ttttctgtca gtatcttacg aatacgaaca gcagcttgcg gatctttcgc taccatatat
      421 aagtacccgc caccaccggc tcccggcaat ttatatccca aggtataatc ttttatcaga
      481 tcgataattg cctccacagc cggaggattt gttccgctat ccaatgcttt gttttgttcc
      541 caggtttttc ctaccaaacg gccaaactca acaaaacttc cacgctgtat agcttcattc
      601 atatccaatg catgcgcctt catttccgaa agtaaattga gatgcaacga tgaattgagg
      661 aacatggaac tgactatttc tgccaagatc ccttttgccg tacgagttat accggtataa
      721 taaagcaagt gacagtcttt gtattcagga tgcgtaaata aatgatcggg tagccaacgc
      781 accaatggac tttgagcaaa gccggcctcg gtctgaagaa gcttcacacc ctgcaacaca
      841 cctccatact gatcctgcca tccacctccg gtagtcagca attgttcaag aacaagagta
      901 cgttgacaaa tctcattttt atcccaggct aaaccacaga aatcgttaat ggcaccaagt
      961 acggtagaag ccagaatgga actggtgccc aaaccggaac cggcaggaat agcagccaat
     1021 aaagtcactt caatacctgc accgaaatct ttaagctgtt cctctaatga agcataagat
     1081 acagcagaaa acgcaggtgc aaagcctgcc aatgacagag cggctttcgg aatagagaaa
     1141 ggtgaaccga tcttcttata atcttgcaat tcatcaaacg tagatactat ttccatagca
     1201 cccatatcga tagaacgcag gacgatatgg aagtctttac acggcttcac atagacctgt
     1261 aagggaggtt gtccgttcaa ctcaatggct agattcacca catttcctcc cgaataaagt
     1321 gaataaggag gagtatcggt ccatccacct gccatatcga tgcgcacggg gctacgtccc
     1381 caaacaatct gatcggaata tacatccaat tttggggtac tcttacgatt actgatcccg
     1441 tccagcaagc cgtcacgaag caaatcaaaa gcagcctgtt cttccggacg ataatctttc
     1501 ccgtctaatt tcaaaatacg cgcacgcaac atccggttat ggatgcgtga catctgcaga
     1561 gcatcctcag gcaataattc aggcatatcc aaaccaagac gtacaaaatc ttcagctgca
     1621 tcggccaaat caagttgata aaaaacactt ttttcatgat taacggccaa tgctttccag
     1681 tttccttttc tgaactcttc acgttgtgca tacaaacgct tcagattggc acctgccgaa
     1741 atttcgtccg cagaaaaatg ttcggaacgt aaccagatat tctttccttc ctccagttcg
     1801 ggttcggaca acatccacct caacaccaat cccaactctt ctacagaatt aaccataggg
     1861 aatacggaaa ctgcctgtaa atcgtccgta cgtcctttca aatctgtata tgacaaaccg
     1921 cgtttggaca tccattcacc aaaaggaata cccgtcaggg ttgttttgga atctctcaaa
     1981 tctcctttga aaacatcgtc cagaccgtat ggacgggcaa caaagccctt atcacccatc
     2041 ggaaccacat ctacacacac tccggcaggc acagccaatg accagtcatt ttccggaacc
     2101 ccggtaataa tatgtcgtga agcaatcttc cactttggtc cgatatgact gttctcgatc
     2161 cataaatcag cattctcggc acaaagaggt atccgcacga cagcattttg gacaaacata
     2221 gccggattgg gctttacttt acggtgcatg atacgacgct gatcgtaaac cttattctgt
     2281 acggaaagag ttgaagaaat cagttcttta ctggtcccgt aatgatagaa ctctcctccc
     2341 ggcaaaggca gaatagcaac ggatagcgta ttgacctctt cgtcttcaat acggggatga
     2401 gttcccaaag ctaatccaaa atcggaataa agatcataat actttagttc ttcagagctt
     2461 tctttatgag aacgtttcat caagatttct acggcacggt cactcaaaag ccatataccg
     2521 atgtccatca ggaacaaatg ggtcttcgat aaagattcca attctgccaa cgaaggcttc
     2581 tgaagcataa agtcgagttg ttcgggatgt ttgcgatcgg aagcaaacac gccatgatgg
     2641 gtagccagag acggatctac ccacagtcca taacaaacca catccgcttc gggaatactc
     2701 tgcaaaggtt tctccgaacg aatatagaca tcaccactcg caatcagtgt atggagttta
     2761 tccggagcca aagacatgat tttttcatat aggggaagtt gcagagaaag cagattttgt
     2821 cccagatgtt gccctctctc ccaccggaac acaggaaccg gagtgagaat ctttccagaa
     2881 ggtgcatagc cgggtaaacg acggctttgc ccacccgcat gaagaagaat tcttttttct
     2941 ttttcaagcc actctccaaa agtagcacca tctgaatatt cattataaca ttcttcaagc
     3001 agccaggatg ttccaccacc ggaaccaagt ttcttaccta ccgggtcgga agtacaaaac
     3061 caatcggtac gattcaccct ctccagttca tgaaaagact gaaccagatt ggacggtaaa
     3121 gatagtagtt tttgggtacc gcccatggta tatctccttc ttaaagttaa acaaaattat
     3181 tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg
     3241 ggatcgagat cgatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc
     3301 acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc
     3361 cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
     3421 ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc
     3481 ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat
     3541 cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     3601 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     3661 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     3721 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     3781 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     3841 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     3901 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     3961 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     4021 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     4081 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     4141 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     4201 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     4261 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     4321 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     4381 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gacatctcgg tagtgggata
     4441 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     4501 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     4561 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     4621 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     4681 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     4741 aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     4801 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     4861 taggacaggt gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg
     4921 cgacgatgat cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct
     4981 tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg
     5041 cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg
     5101 gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct
     5161 gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt
     5221 aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag
     5281 gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac
     5341 cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac
     5401 gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt
     5461 tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca
     5521 aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc
     5581 tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg
     5641 accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
     5701 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     5761 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
     5821 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt
     5881 actgagagtg caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     5941 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     6001 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     6061 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     6121 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     6181 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     6241 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     6301 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     6361 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     6421 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6481 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6541 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     6601 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6661 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6721 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6781 gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     6841 tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga
     6901 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     6961 ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     7021 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     7081 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     7141 gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     7201 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     7261 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     7321 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     7381 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     7441 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     7501 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     7561 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     7621 attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca
     7681 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7741 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     7801 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     7861 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     7921 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     7981 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     8041 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     8101 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     8161 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg
     8221 gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt
     8281 tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg ggctttgtta
     8341 gcagccggat ctcagtggtg gtggtggtgg tgctcgagtg cggccgcaag cttcatatgc
     8401 tcgagggagc caccagtctc cggtaaagaa ccgctatcca tgtgattgcg ctcaaaacgg
     8461 gctgctgcgg tttcacgaga tctgctgctc acctgcgcaa cacccgggac gccaacgccc
     8521 ggaacgccca cacccgggac gccaacgccc ggaacgccca cacccgggac gccaacgccc
     8581 ggaacgccca cacccgggac gccaacgccc ggaacgccca cacccgggac gccaacgccc
     8641 ggaacgccca cacccgggac gccaacgccc ggaacgccca cacccgggac gccaacgccc
     8701 ggaacgccca cacccgggac gccaacgccc ggaacgccca cacccg
//
