LOCUS       sequence-298545-        9050 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of biosynthetic enzymes 4CL and STS in fusion with CC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9050)
  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 9050)
  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:P7SHb + STS:P8SHb
FEATURES             Location/Qualifiers
     source          1..9050
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(27..74)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1426..1440)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(1456..1485)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1498..1520
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1535..1559
                     /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)."
     promoter        complement(1560..1578)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(1763..1810)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3672..3686)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(3702..3731)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             3744..3766
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3781..3805
                     /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)."
     promoter        complement(3806..3824)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3908..3927)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4133..4210
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4211..5293
                     /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(4230..4249)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6605..6796
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6790..6812)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6898..7037
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6953..6972
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(7052..7069)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7223..7811)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7303..7322)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7982..8773,8774..8842))
                     /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     8605..8624
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8843..8947)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     9015..9033
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttcccggata tagttcctcc tttcagcaaa aaacccctca agacccgttt agaggcccca
       61 aggggttatg ctagttattg ctcagcggtg gcagcagcca actcagcttc ctttcgggct
      121 ttgttagcag ccggatcctt agcgctcaag atactcgttc tcgcgcttta attcgcggat
      181 tttgcgctcc aattcgcggt tttcgcgctt cagctcttcg attttatctt cgggggagcc
      241 gctgccattg gtaaccatcg gaatgctatg cagaacaacg gtttcaatgg tcagacccgg
      301 accaaaacca aacagaacac cccaatccag accttcaccg gtggttgcac gttcaccttt
      361 caggcttttt ttgcgcattt catccagaat aaacagaaca catgcgctgc tcatattacc
      421 atattcgctc agaacatgac gggttgcttc cagtttcttt ttgtccaggt tcagttttgc
      481 ttcaactgca tccagaattg ccggaccacc cggatgtgca atccaaaaca ggctattcca
      541 atcgctaata cccagcggat caaatgcctg ggtcagacat ttttcaacat tttcgctaat
      601 cagggtcgga acattcggcc acagatgaaa ggtcagacca acttcacgca gattacctgc
      661 aattgcaccg gcactattcg gaataaaggt ctgtgctgcg ctaaccagct gaaacagcgg
      721 acgttcaatg ctaatatccg gatcgctacc aacaataact gctgcgctac catcaccaaa
      781 cagtgcctga ccaaccaggc tatccagtgc atcttcgctc ggaccacgaa aggtaacaac
      841 ggtaatttcg ctacaaacaa ccagaacacg tgcaccggca ttattttctg ccagatcttt
      901 tgcggtacgc agaacggtgc caccggcata acaaccctga tgatacagca taacacgacg
      961 aacgctcggt tccagaccca gcagatttgc cagtttataa tctgcaccag gcatttcaac
     1021 accactggtg gtacaaaaaa ccagatgggt aattttgctt ttcggctgac cccattcttt
     1081 cagggctttc agtgctgctt ctttacccag tttcggaact tctgcggtga taatttcttg
     1141 acgaatattc aggctcggtg ccatatatgc accaatattc ggatgttctt ccagcatttc
     1201 ttcggtcagg tggatatagc gttttttgat catgctttta tcgcagatgc ggttaaattt
     1261 ctttttcagt gcggtcatgt gttcgctttt ggtcacttta aagtaataat cggcataatc
     1321 gctctgataa acacaatgat ccggtgttgc ggtgccaatt gccagaatgg ttgccggacc
     1381 ttttgcacgc tgtgcattac gaaattcttc aacgcttgcc atatgcttgt cgtcgtcgtc
     1441 gatatggccg ctgctgtgat gatgatgatg atgatgatga tgatggccca tggtatatct
     1501 ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc
     1561 ctatagtgag tcgtattaaa gctttaatgc ggtagtttat cacagttaaa ttgctaacgc
     1621 agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg caccgtcacc
     1681 ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt gcgggatatc
     1741 cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag gccccaaggg
     1801 gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt cgggctttgt
     1861 tagcagccgg atccttaacg tttcaaatac tcgttctttt cctccagctc acggatttcg
     1921 cgtttaagct cttcattttt ttcttccaat tcacgaatct cgtcttccgg ggagccgctg
     1981 ccacccagac catttgccag ttttgcacgc agatctttac gcagaatttt accgctcggt
     2041 gctttcggaa tgctctcggt aaaaaacact ttgttgatgc gtttgtagaa caccacctgt
     2101 ttgctaacaa actgtttaac atcatcttcg ctcagttcgc tatctttgct tttcacaaca
     2161 aatgcaaccg gaacttcgcc tgctgcttct tctttcattg caacaactgc aacatcggta
     2221 atatccggat gaccaatcag cagtgcttcc agttctgccg gtgcaacctg aaaacctttg
     2281 tatttgatca gctctttcag acgatccaca ataaacagtt cgtcatcatc atcaatcaga
     2341 ccaatatcac ctgtatgcag ccaaccatct ttatcaatgg tttctgcggt tgctgccgga
     2401 ttattcagat agcctttcat aatctggtga ccacgaatac aaatttcacc aggctgatta
     2461 cggctcaggc tatcaccggt atccggatca acaattttca tttctgcatt acgaacaacg
     2521 gtgccacatg caccactttt aaccggaaac ggttcttttg caaaacccag gctcattgcc
     2581 agaaccggac ctgcttcggt cataccataa ccctgaccca gttttgcatt cggaaatttg
     2641 gcattaactg catcttccag ttctttaccc agcggagctg caccgctttt aacaacacga
     2701 atgctgctca gatcatattt ttcggtttcg ctgctttttg caattgccag aacaataggc
     2761 ggaaccatcg gtgcaacggt aactttacaa cgctggatca gttccagcag cagattaatt
     2821 tcaaatttcg gcataatcag aattgctgca ccaacacgca gaccacacag cataatgcta
     2881 ttcagtgcat aaatatgaaa catcggcagc acacacagaa taacatcgtc gctatgaaaa
     2941 tacagattcg gattttcacc atcaacctgc tgtgcaacgc tggtaaccag acctttatgg
     3001 gtcagcataa cacctttcgg cagaccggtg gtgccgctgc tatacggcag tgcaacaaca
     3061 tcatccggac taatttcaac gctatcaata acttcgcttg cttcggtggt gctctgggtc
     3121 agttcggtaa aacgcagaca accttccgga atcggaacgc tttcattatc atcgatgcaa
     3181 acaatcacaa caccatcatc attttgcagc ggtttgattt tgtccacgta acgtgcttcg
     3241 gtaataatca gtttggtgtt gcttgctttt gcctgttttg caatttctgc cggtgtaaaa
     3301 aacggatttg ctgcggttgc ggttgcacca cgaaagctgg ctgccagaaa gctcagaaca
     3361 aattccggac aattcggcag cagcagcata acaacgtcat tctgattcac acccagttta
     3421 tgaaaattgg ctgcaatctg acggctgatc acatgaacat cactataggt ataaacatga
     3481 ccggtcggac cattaatcag acacggtttg gtggcaaact cgctaatgtt ctgaaagata
     3541 taatcgtgca ggctcagatg attcggaata tagatatccg gcagtttgct acgaaaaatc
     3601 acgtcgctgt tgttattgtt gctctgtttt tccataacct ggctaactgc ctgttcttgc
     3661 ggtgccatat gcttgtcgtc gtcgtcgata tggccgctgc tgtgatgatg atgatgatga
     3721 tgatgatgat ggcccatggt atatctcctt cttaaagtta aacaaaatta tttctagagg
     3781 ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag
     3841 atctcgatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt
     3901 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc
     3961 atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc
     4021 gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
     4081 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat
     4141 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     4201 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     4261 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     4321 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     4381 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     4441 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     4501 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     4561 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     4621 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     4681 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     4741 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     4801 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     4861 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     4921 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     4981 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     5041 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     5101 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     5161 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     5221 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     5281 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat
     5341 ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     5401 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     5461 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
     5521 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
     5581 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc
     5641 tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc
     5701 ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag
     5761 atgacgacca tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga
     5821 tcattggacc gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt
     5881 tggcatggat tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg
     5941 catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac
     6001 cactccaaga attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc
     6061 ttggcagaac atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg
     6121 cagcgttggg tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct
     6181 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg
     6241 aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt
     6301 ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat
     6361 ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg
     6421 ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt
     6481 accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat
     6541 cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc
     6601 atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac
     6661 attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga
     6721 atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga
     6781 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     6841 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     6901 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     6961 gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga
     7021 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     7081 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     7141 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     7201 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     7261 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     7321 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     7381 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     7441 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     7501 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     7561 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     7621 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     7681 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     7741 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     7801 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     7861 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     7921 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     7981 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     8041 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     8101 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     8161 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     8221 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     8281 acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     8341 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     8401 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     8461 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     8521 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     8581 gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     8641 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     8701 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     8761 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     8821 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     8881 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     8941 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     9001 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa
//
