LOCUS       sequence-298541-        9065 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 9065)
  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 9065)
  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:P3S + STS:AP4S
FEATURES             Location/Qualifiers
     source          1..9065
                     /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(1432..1446)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(1462..1491)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1504..1526
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1541..1565
                     /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(1566..1584)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(1769..1816)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3687..3701)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(3717..3746)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             3759..3781
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3796..3820
                     /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(3821..3839)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3923..3942)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4148..4225
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4226..5308
                     /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(4245..4264)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6620..6811
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6805..6827)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6913..7052
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6968..6987
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(7067..7084)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7238..7826)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7318..7337)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7997..8788,8789..8857))
                     /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     8620..8639
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8858..8962)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     9030..9048
                     /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 agccgtattt aatactctgc aacttttgct tgatttgttg
      181 taatttctgt tcattttgtt gcagttcctc ttcgttggat tgaagctcgt cctctggcga
      241 gctgccgctg ccattggtaa ccatcggaat gctatgcaga acaacggttt caatggtcag
      301 acccggacca aaaccaaaca gaacacccca atccagacct tcaccggtgg ttgcacgttc
      361 acctttcagg ctttttttgc gcatttcatc cagaataaac agaacacatg cgctgctcat
      421 attaccatat tcgctcagaa catgacgggt tgcttccagt ttctttttgt ccaggttcag
      481 ttttgcttca actgcatcca gaattgccgg accacccgga tgtgcaatcc aaaacaggct
      541 attccaatcg ctaataccca gcggatcaaa tgcctgggtc agacattttt caacattttc
      601 gctaatcagg gtcggaacat tcggccacag atgaaaggtc agaccaactt cacgcagatt
      661 acctgcaatt gcaccggcac tattcggaat aaaggtctgt gctgcgctaa ccagctgaaa
      721 cagcggacgt tcaatgctaa tatccggatc gctaccaaca ataactgctg cgctaccatc
      781 accaaacagt gcctgaccaa ccaggctatc cagtgcatct tcgctcggac cacgaaaggt
      841 aacaacggta atttcgctac aaacaaccag aacacgtgca ccggcattat tttctgccag
      901 atcttttgcg gtacgcagaa cggtgccacc ggcataacaa ccctgatgat acagcataac
      961 acgacgaacg ctcggttcca gacccagcag atttgccagt ttataatctg caccaggcat
     1021 ttcaacacca ctggtggtac aaaaaaccag atgggtaatt ttgcttttcg gctgacccca
     1081 ttctttcagg gctttcagtg ctgcttcttt acccagtttc ggaacttctg cggtgataat
     1141 ttcttgacga atattcaggc tcggtgccat atatgcacca atattcggat gttcttccag
     1201 catttcttcg gtcaggtgga tatagcgttt tttgatcatg cttttatcgc agatgcggtt
     1261 aaatttcttt ttcagtgcgg tcatgtgttc gcttttggtc actttaaagt aataatcggc
     1321 ataatcgctc tgataaacac aatgatccgg tgttgcggtg ccaattgcca gaatggttgc
     1381 cggacctttt gcacgctgtg cattacgaaa ttcttcaacg cttgccatat gcttgtcgtc
     1441 gtcgtcgata tggccgctgc tgtgatgatg atgatgatga tgatgatgat ggcccatggt
     1501 atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca
     1561 attcccctat agtgagtcgt attaaagctt taatgcggta gtttatcaca gttaaattgc
     1621 taacgcagtc aggcaccgtg tatgaaatct aacaatgcgc tcatcgtcat cctcggcacc
     1681 gtcaccctgg atgctgtagg cataggcttg gttatgccgg tactgccggg cctcttgcgg
     1741 gatatccgga tatagttcct cctttcagca aaaaacccct caagacccgt ttagaggccc
     1801 caaggggtta tgctagttat tgctcagcgg tggcagcagc caactcagct tcctttcggg
     1861 ctttgttagc agccggatcc ttaatagcca tatttcagtt cctggttttt ttctttcagt
     1921 tcgctgtttt tctgttccag ctggctaatt tcttcttcca gctgctgaat ttcatcttcc
     1981 gggctgctgc cgctgccacc cagaccattt gccagttttg cacgcagatc tttacgcaga
     2041 attttaccgc tcggtgcttt cggaatgctc tcggtaaaaa acactttgtt gatgcgtttg
     2101 tagaacacca cctgtttgct aacaaactgt ttaacatcat cttcgctcag ttcgctatct
     2161 ttgcttttca caacaaatgc aaccggaact tcgcctgctg cttcttcttt cattgcaaca
     2221 actgcaacat cggtaatatc cggatgacca atcagcagtg cttccagttc tgccggtgca
     2281 acctgaaaac ctttgtattt gatcagctct ttcagacgat ccacaataaa cagttcgtca
     2341 tcatcatcaa tcagaccaat atcacctgta tgcagccaac catctttatc aatggtttct
     2401 gcggttgctg ccggattatt cagatagcct ttcataatct ggtgaccacg aatacaaatt
     2461 tcaccaggct gattacggct caggctatca ccggtatccg gatcaacaat tttcatttct
     2521 gcattacgaa caacggtgcc acatgcacca cttttaaccg gaaacggttc ttttgcaaaa
     2581 cccaggctca ttgccagaac cggacctgct tcggtcatac cataaccctg acccagtttt
     2641 gcattcggaa atttggcatt aactgcatct tccagttctt tacccagcgg agctgcaccg
     2701 cttttaacaa cacgaatgct gctcagatca tatttttcgg tttcgctgct ttttgcaatt
     2761 gccagaacaa taggcggaac catcggtgca acggtaactt tacaacgctg gatcagttcc
     2821 agcagcagat taatttcaaa tttcggcata atcagaattg ctgcaccaac acgcagacca
     2881 cacagcataa tgctattcag tgcataaata tgaaacatcg gcagcacaca cagaataaca
     2941 tcgtcgctat gaaaatacag attcggattt tcaccatcaa cctgctgtgc aacgctggta
     3001 accagacctt tatgggtcag cataacacct ttcggcagac cggtggtgcc gctgctatac
     3061 ggcagtgcaa caacatcatc cggactaatt tcaacgctat caataacttc gcttgcttcg
     3121 gtggtgctct gggtcagttc ggtaaaacgc agacaacctt ccggaatcgg aacgctttca
     3181 ttatcatcga tgcaaacaat cacaacacca tcatcatttt gcagcggttt gattttgtcc
     3241 acgtaacgtg cttcggtaat aatcagtttg gtgttgcttg cttttgcctg ttttgcaatt
     3301 tctgccggtg taaaaaacgg atttgctgcg gttgcggttg caccacgaaa gctggctgcc
     3361 agaaagctca gaacaaattc cggacaattc ggcagcagca gcataacaac gtcattctga
     3421 ttcacaccca gtttatgaaa attggctgca atctgacggc tgatcacatg aacatcacta
     3481 taggtataaa catgaccggt cggaccatta atcagacacg gtttggtggc aaactcgcta
     3541 atgttctgaa agatataatc gtgcaggctc agatgattcg gaatatagat atccggcagt
     3601 ttgctacgaa aaatcacgtc gctgttgtta ttgttgctct gtttttccat aacctggcta
     3661 actgcctgtt cttgcggtgc catatgcttg tcgtcgtcgt cgatatggcc gctgctgtga
     3721 tgatgatgat gatgatgatg atgatggccc atggtatatc tccttcttaa agttaaacaa
     3781 aattatttct agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa
     3841 tttcgcggga tcgagatctc gatcctctac gccggacgca tcgtggccgg catcaccggc
     3901 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct
     3961 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc
     4021 gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac
     4081 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga
     4141 gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga
     4201 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     4261 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     4321 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     4381 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     4441 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     4501 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     4561 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     4621 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     4681 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     4741 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     4801 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     4861 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     4921 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     4981 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
     5041 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     5101 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     5161 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     5221 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     5281 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     5341 attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc
     5401 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac
     5461 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga
     5521 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag
     5581 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca
     5641 tggcggccga cgcgctgggc tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct
     5701 tccccattat gattcttctc gcttccggcg gcatcgggat gcccgcgttg caggccatgc
     5761 tgtccaggca ggtagatgac gaccatcagg gacagcttca aggatcgctc gcggctctta
     5821 ccagcctaac ttcgatcatt ggaccgctga tcgtcacggc gatttatgcc gcctcggcga
     5881 gcacatggaa cgggttggca tggattgtag gcgccgccct ataccttgtc tgcctccccg
     5941 cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct
     6001 cgctaacgga ttcaccactc caagaattgg agccaatcaa ttcttgcgga gaactgtgaa
     6061 tgcgcaaacc aacccttggc agaacatatc catcgcgtcc gccatctcca gcagccgcac
     6121 gcggcgcatc tcgggcagcg ttgggtcctg gccacgggtg cgcatgatcg tgctcctgtc
     6181 gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat
     6241 acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg
     6301 aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac
     6361 cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc
     6421 tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca
     6481 taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac
     6541 ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc
     6601 cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt
     6661 tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca
     6721 ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc
     6781 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     6841 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     6901 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     6961 aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt gaaataccgc
     7021 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     7081 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     7141 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     7201 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     7261 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     7321 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     7381 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     7441 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     7501 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     7561 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     7621 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     7681 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     7741 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     7801 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     7861 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     7921 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     7981 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     8041 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     8101 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     8161 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     8221 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     8281 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
     8341 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     8401 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     8461 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     8521 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     8581 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     8641 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     8701 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     8761 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     8821 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     8881 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     8941 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     9001 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     9061 aagaa
//
