LOCUS       sequence-479231-        7354 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  Expression an ELP-tagged fusion enzyme of NanA and CSS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7354)
  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 7354)
  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-NanA-CSS-ET64
FEATURES             Location/Qualifiers
     source          1..7354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1821..1843
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1857..1881
                     /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(1881..1900)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1882..1900)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1988..2007)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2213..2290
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2291..3373
                     /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(2310..2329)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4182..4373
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4367..4389)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4475..4614
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4530..4549
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4629..4646)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4800..5388)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4880..4899)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5499..5518)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5510..6325
                     /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             5510..6325
                     /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(5583..5602)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6417..6872)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6554..6575)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6766..6785
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(6909..6956)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6952..6970
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(7023..7040)
                     /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 acaccggaga ccgtggatct gccgctctct
      241 ttgtggttca gaatattctc ggcttgctgc agatccagtt cggtgtcaat gtcgatgcta
      301 tcttggtggc tcatgatgta cagcttggtc ggcgcgatga agaagcagtt gttggcgatc
      361 agactggccg tgtcgttaat gtagatggca ccgttcgggc gaaaggcttg tggcagctgc
      421 tgacgcggct gttccagatc gctcagatgg cgcatcggcg cgtactcacc gttgttgatc
      481 tggaggagcg ttttcagcgg atgatgttcc atcgggcacg cgctaaccac gctgcccttg
      541 attttctcgt cgaacagact gaacgcttcg cggatatgcg caccggtgcg cagtggactg
      601 gttggctgca gcagcgtaac ggtgccgcta ttgctaccga tggtttccag cgcatggata
      661 acaccactga tgctgcgggc ggtatcactc gccagttccg ctggacggag caccacttcc
      721 acgccgaaat tcttcgcctc ttcggcgatg agaccgccat cggtgctcac aatgatgcgg
      781 tcgaagcact tgctgctgat cgcggcgtta atggtatggc ccagcagact gatgccattc
      841 atcttgcgca gattcttcag cgggagacct ttgctgttct ggcgggccag aatcaccgca
      901 atgttctgtt tctcggtacc gctagatcta ccgctcagat atttggcctt gagctctttg
      961 gcgaaggcaa ccttttcgct gctcagctcc ttggtcatcg gttcgcgaca atagcccgct
     1021 tccacaccgt cgagcttcag cagctctttg atggtcagat acagaccatt ggccagaatg
     1081 ccttcgatca gatcgttggt cacgtgctgg atctccagcg cctctttcag tttgccggct
     1141 tgcgtcagtt cgaagatttg gcgggcgcga acgccgttca cgttgaaggt actaccgatc
     1201 gcaccatcca cacccagact cgcggctggc agcatcattt catcaaagcc ggcccagatg
     1261 aggtggtttg ggtacgcctt tttcagacgc tccagcagat agaaatcgcc ggcggtgaat
     1321 ttcacgccga gcacctttgg gttcttgtac agctcgccga actgttcaac gccgatattc
     1381 acgccggtca gaaacgggat gctgtacacg atcatgtagt tgccggtggc ctcgatgata
     1441 ctgtcatagt agtgtttgat ttccgggaag ctaaacttat aatagaatgg ggtcacggcg
     1501 ctcagactat cgtagcccag ttcggtcgca tatttgccca gttcgatggc ttcttgcaga
     1561 ttaacgctgc caacttgcgc gatcagcgcg atttcgtctt tcgcctcatc cttggcgatg
     1621 cggaagattt ccttcttctc ctcggtgctg agcatgaaat tctcgccggt gctgccgcca
     1681 acatagaggc catccacctt catcttgtcg atgttgtaac gcacgatctg gcgcagaccc
     1741 ttctcgttga tgctgccatc cgcattgaag ctaaccagca gcgcgctgaa gatgcccttc
     1801 agatttttgc ccatggtata tctccttctt aaagttaaac aaaattattc tagaggggaa
     1861 ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatcg
     1921 atctcgatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt
     1981 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc
     2041 atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc
     2101 gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
     2161 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat
     2221 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     2281 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     2341 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     2401 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     2461 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     2521 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     2581 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     2641 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     2701 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     2761 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     2821 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     2881 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     2941 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     3001 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     3061 taccgagtcc gggctgcgcg ttggtgcgga catctcggta gtgggatacg acgataccga
     3121 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     3181 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     3241 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     3301 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     3361 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat
     3421 ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     3481 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     3541 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
     3601 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
     3661 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg
     3721 tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg
     3781 gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt
     3841 ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa
     3901 cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc
     3961 taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt
     4021 ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc
     4081 gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca
     4141 ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa
     4201 ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca
     4261 acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg
     4321 agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     4381 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     4441 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     4501 atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     4561 ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
     4621 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4681 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4741 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4801 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4861 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4921 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4981 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5041 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5101 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5161 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5221 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5281 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5341 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5401 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5461 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
     5521 tcaacgggaa acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata
     5581 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta
     5641 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     5701 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     5761 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa
     5821 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     5881 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     5941 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     6001 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     6061 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     6121 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     6181 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     6241 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     6301 tttgatgctc gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta
     6361 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     6421 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     6481 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     6541 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     6601 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     6661 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     6721 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     6781 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     6841 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccaatccgga tatagttcct
     6901 cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat
     6961 tgctcagcgg tggcagcagc caactcagct tcctttcggg ctttgttagc agccggatct
     7021 cagtggtggt ggtggtggtg ctcgagtgcg gccgcaagct tcatatgctc gagggagcca
     7081 ccagtctccg gtaaagaacc gctatccatg tgattgcgct caaaacgggc tgctgcggtt
     7141 tcacgagatc tgctgctcac ctgcgcaaca cccgggacgc caacgcccgg aacgcccaca
     7201 cccgggacgc caacgcccgg aacgcccaca cccgggacgc caacgcccgg aacgcccaca
     7261 cccgggacgc caacgcccgg aacgcccaca cccgggacgc caacgcccgg aacgcccaca
     7321 cccgggacgc caacgcccgg aacgcccaca cccg
//
