LOCUS       sequence-298688-        8601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  direct fusion of biosynthetic enzymes 4CL and STS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8601)
  AUTHORS   Fink T, Stevovic B, Verwaal R, Roubos JA, Gaber R, Bencina M, Jerala
            R, Gradisar H
  TITLE     Metabolic enzyme clustering by coiled coils improves the 
            biosynthesis of resveratrol and mevalonate.
  JOURNAL   AMB Express. 2020 May 24;10(1):97. doi: 10.1186/s13568-020-01031-5.
  PUBMED    32448937
REFERENCE   2  (bases 1 to 8601)
  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: "AMB 
            Express."; date: "2020-05-24"; volume: "10(1):97. doi"; pages: " 
            10.1186/s13568-020-01031-5"
COMMENT     Sequence Label: pET19b_4CL:STS (DF)
FEATURES             Location/Qualifiers
     source          1..8601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(213..260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     256..274
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3223..3237)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(3253..3282)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             3295..3317
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3332..3356
                     /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(3356..3375)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3357..3375)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3459..3478)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3684..3761
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3762..4844
                     /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(3781..3800)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6156..6347
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6341..6363)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6449..6588
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6504..6523
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6603..6620)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6774..7362)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6854..6873)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7533..8324,8325..8393))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8156..8175
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8394..8498)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8566..8584
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
      241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
      301 cgggctttgt tagcagccgg atccattggt aaccatcgga atgctatgca gaacaacggt
      361 ttcaatggtc agacccggac caaaaccaaa cagaacaccc caatccagac cttcaccggt
      421 ggttgcacgt tcacctttca ggcttttttt gcgcatttca tccagaataa acagaacaca
      481 tgcgctgctc atattaccat attcgctcag aacatgacgg gttgcttcca gtttcttttt
      541 gtccaggttc agttttgctt caactgcatc cagaattgcc ggaccacccg gatgtgcaat
      601 ccaaaacagg ctattccaat cgctaatacc cagcggatca aatgcctggg tcagacattt
      661 ttcaacattt tcgctaatca gggtcggaac attcggccac agatgaaagg tcagaccaac
      721 ttcacgcaga ttacctgcaa ttgcaccggc actattcgga ataaaggtct gtgctgcgct
      781 aaccagctga aacagcggac gttcaatgct aatatccgga tcgctaccaa caataactgc
      841 tgcgctacca tcaccaaaca gtgcctgacc aaccaggcta tccagtgcat cttcgctcgg
      901 accacgaaag gtaacaacgg taatttcgct acaaacaacc agaacacgtg caccggcatt
      961 attttctgcc agatcttttg cggtacgcag aacggtgcca ccggcataac aaccctgatg
     1021 atacagcata acacgacgaa cgctcggttc cagacccagc agatttgcca gtttataatc
     1081 tgcaccaggc atttcaacac cactggtggt acaaaaaacc agatgggtaa ttttgctttt
     1141 cggctgaccc cattctttca gggctttcag tgctgcttct ttacccagtt tcggaacttc
     1201 tgcggtgata atttcttgac gaatattcag gctcggtgcc atatatgcac caatattcgg
     1261 atgttcttcc agcatttctt cggtcaggtg gatatagcgt tttttgatca tgcttttatc
     1321 gcagatgcgg ttaaatttct ttttcagtgc ggtcatgtgt tcgcttttgg tcactttaaa
     1381 gtaataatcg gcataatcgc tctgataaac acaatgatcc ggtgttgcgg tgccaattgc
     1441 cagaatggtt gccggacctt ttgcacgctg tgcattacga aattcttcaa cgcttgcgga
     1501 gccgccggag ccggagccgg agcccccacc cagaccattt gccagttttg cacgcagatc
     1561 tttacgcaga attttaccgc tcggtgcttt cggaatgctc tcggtaaaaa acactttgtt
     1621 gatgcgtttg tagaacacca cctgtttgct aacaaactgt ttaacatcat cttcgctcag
     1681 ttcgctatct ttgcttttca caacaaatgc aaccggaact tcgcctgctg cttcttcttt
     1741 cattgcaaca actgcaacat cggtaatatc cggatgacca atcagcagtg cttccagttc
     1801 tgccggtgca acctgaaaac ctttgtattt gatcagctct ttcagacgat ccacaataaa
     1861 cagttcgtca tcatcatcaa tcagaccaat atcacctgta tgcagccaac catctttatc
     1921 aatggtttct gcggttgctg ccggattatt cagatagcct ttcataatct ggtgaccacg
     1981 aatacaaatt tcaccaggct gattacggct caggctatca ccggtatccg gatcaacaat
     2041 tttcatttct gcattacgaa caacggtgcc acatgcacca cttttaaccg gaaacggttc
     2101 ttttgcaaaa cccaggctca ttgccagaac cggacctgct tcggtcatac cataaccctg
     2161 acccagtttt gcattcggaa atttggcatt aactgcatct tccagttctt tacccagcgg
     2221 agctgcaccg cttttaacaa cacgaatgct gctcagatca tatttttcgg tttcgctgct
     2281 ttttgcaatt gccagaacaa taggcggaac catcggtgca acggtaactt tacaacgctg
     2341 gatcagttcc agcagcagat taatttcaaa tttcggcata atcagaattg ctgcaccaac
     2401 acgcagacca cacagcataa tgctattcag tgcataaata tgaaacatcg gcagcacaca
     2461 cagaataaca tcgtcgctat gaaaatacag attcggattt tcaccatcaa cctgctgtgc
     2521 aacgctggta accagacctt tatgggtcag cataacacct ttcggcagac cggtggtgcc
     2581 gctgctatac ggcagtgcaa caacatcatc cggactaatt tcaacgctat caataacttc
     2641 gcttgcttcg gtggtgctct gggtcagttc ggtaaaacgc agacaacctt ccggaatcgg
     2701 aacgctttca ttatcatcga tgcaaacaat cacaacacca tcatcatttt gcagcggttt
     2761 gattttgtcc acgtaacgtg cttcggtaat aatcagtttg gtgttgcttg cttttgcctg
     2821 ttttgcaatt tctgccggtg taaaaaacgg atttgctgcg gttgcggttg caccacgaaa
     2881 gctggctgcc agaaagctca gaacaaattc cggacaattc ggcagcagca gcataacaac
     2941 gtcattctga ttcacaccca gtttatgaaa attggctgca atctgacggc tgatcacatg
     3001 aacatcacta taggtataaa catgaccggt cggaccatta atcagacacg gtttggtggc
     3061 aaactcgcta atgttctgaa agatataatc gtgcaggctc agatgattcg gaatatagat
     3121 atccggcagt ttgctacgaa aaatcacgtc gctgttgtta ttgttgctct gtttttccat
     3181 aacctggcta actgcctgtt cttgcggtgc ctcgagcata tgcttgtcgt cgtcgtcgat
     3241 atggccgctg ctgtgatgat gatgatgatg atgatgatga tggcccatgg tatatctcct
     3301 tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta
     3361 tagtgagtcg tattaatttc gcgggatcga gatctcgatc ctctacgccg gacgcatcgt
     3421 ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga
     3481 tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt
     3541 ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc
     3601 ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca
     3661 taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg
     3721 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     3781 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     3841 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     3901 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     3961 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     4021 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     4081 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     4141 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     4201 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     4261 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     4321 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     4381 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     4441 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     4501 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     4561 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     4621 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     4681 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     4741 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     4801 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     4861 gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac
     4921 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc
     4981 ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag
     5041 gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg
     5101 cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag
     5161 gccattatcg ccggcatggc ggccgacgcg ctgggctacg tcttgctggc gttcgcgacg
     5221 cgaggctgga tggccttccc cattatgatt cttctcgctt ccggcggcat cgggatgccc
     5281 gcgttgcagg ccatgctgtc caggcaggta gatgacgacc atcagggaca gcttcaagga
     5341 tcgctcgcgg ctcttaccag cctaacttcg atcattggac cgctgatcgt cacggcgatt
     5401 tatgccgcct cggcgagcac atggaacggg ttggcatgga ttgtaggcgc cgccctatac
     5461 cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg
     5521 gaagccggcg gcacctcgct aacggattca ccactccaag aattggagcc aatcaattct
     5581 tgcggagaac tgtgaatgcg caaaccaacc cttggcagaa catatccatc gcgtccgcca
     5641 tctccagcag ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca
     5701 tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc
     5761 agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga
     5821 cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga
     5881 agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct
     5941 gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct
     6001 ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat
     6061 gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc
     6121 ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc
     6181 ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc
     6241 tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt
     6301 accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     6361 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     6421 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
     6481 gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccattg
     6541 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
     6601 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     6661 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     6721 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     6781 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     6841 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6901 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6961 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     7021 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     7081 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     7141 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     7201 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     7261 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     7321 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     7381 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     7441 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     7501 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     7561 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     7621 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagagcca
     7681 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     7741 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     7801 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg
     7861 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7921 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7981 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     8041 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     8101 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat
     8161 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     8221 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     8281 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     8341 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     8401 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     8461 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     8521 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     8581 aggccctttc gtcttcaaga a
//
