LOCUS       sequence-298690-        7401 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses biosynthetic enzyme 4CL.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7401)
  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 7401)
  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
FEATURES             Location/Qualifiers
     source          1..7401
                     /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(2023..2037)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(2053..2082)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             2095..2117
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2132..2156
                     /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(2156..2175)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2157..2175)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2259..2278)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2484..2561
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2562..3644
                     /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(2581..2600)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4956..5147
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5141..5163)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5249..5388
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5304..5323
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5403..5420)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5574..6162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5654..5673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6333..7124,7125..7193))
                     /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     6956..6975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7194..7298)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7366..7384
                     /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 atccttaacc cagaccattt gccagttttg cacgcagatc
      361 tttacgcaga attttaccgc tcggtgcttt cggaatgctc tcggtaaaaa acactttgtt
      421 gatgcgtttg tagaacacca cctgtttgct aacaaactgt ttaacatcat cttcgctcag
      481 ttcgctatct ttgcttttca caacaaatgc aaccggaact tcgcctgctg cttcttcttt
      541 cattgcaaca actgcaacat cggtaatatc cggatgacca atcagcagtg cttccagttc
      601 tgccggtgca acctgaaaac ctttgtattt gatcagctct ttcagacgat ccacaataaa
      661 cagttcgtca tcatcatcaa tcagaccaat atcacctgta tgcagccaac catctttatc
      721 aatggtttct gcggttgctg ccggattatt cagatagcct ttcataatct ggtgaccacg
      781 aatacaaatt tcaccaggct gattacggct caggctatca ccggtatccg gatcaacaat
      841 tttcatttct gcattacgaa caacggtgcc acatgcacca cttttaaccg gaaacggttc
      901 ttttgcaaaa cccaggctca ttgccagaac cggacctgct tcggtcatac cataaccctg
      961 acccagtttt gcattcggaa atttggcatt aactgcatct tccagttctt tacccagcgg
     1021 agctgcaccg cttttaacaa cacgaatgct gctcagatca tatttttcgg tttcgctgct
     1081 ttttgcaatt gccagaacaa taggcggaac catcggtgca acggtaactt tacaacgctg
     1141 gatcagttcc agcagcagat taatttcaaa tttcggcata atcagaattg ctgcaccaac
     1201 acgcagacca cacagcataa tgctattcag tgcataaata tgaaacatcg gcagcacaca
     1261 cagaataaca tcgtcgctat gaaaatacag attcggattt tcaccatcaa cctgctgtgc
     1321 aacgctggta accagacctt tatgggtcag cataacacct ttcggcagac cggtggtgcc
     1381 gctgctatac ggcagtgcaa caacatcatc cggactaatt tcaacgctat caataacttc
     1441 gcttgcttcg gtggtgctct gggtcagttc ggtaaaacgc agacaacctt ccggaatcgg
     1501 aacgctttca ttatcatcga tgcaaacaat cacaacacca tcatcatttt gcagcggttt
     1561 gattttgtcc acgtaacgtg cttcggtaat aatcagtttg gtgttgcttg cttttgcctg
     1621 ttttgcaatt tctgccggtg taaaaaacgg atttgctgcg gttgcggttg caccacgaaa
     1681 gctggctgcc agaaagctca gaacaaattc cggacaattc ggcagcagca gcataacaac
     1741 gtcattctga ttcacaccca gtttatgaaa attggctgca atctgacggc tgatcacatg
     1801 aacatcacta taggtataaa catgaccggt cggaccatta atcagacacg gtttggtggc
     1861 aaactcgcta atgttctgaa agatataatc gtgcaggctc agatgattcg gaatatagat
     1921 atccggcagt ttgctacgaa aaatcacgtc gctgttgtta ttgttgctct gtttttccat
     1981 aacctggcta actgcctgtt cttgcggtgc ctcgagcata tgcttgtcgt cgtcgtcgat
     2041 atggccgctg ctgtgatgat gatgatgatg atgatgatga tggcccatgg tatatctcct
     2101 tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta
     2161 tagtgagtcg tattaatttc gcgggatcga gatctcgatc ctctacgccg gacgcatcgt
     2221 ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga
     2281 tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt
     2341 ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc
     2401 ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca
     2461 taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg
     2521 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     2581 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     2641 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     2701 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     2761 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     2821 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     2881 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     2941 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     3001 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     3061 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     3121 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     3181 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     3241 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     3301 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     3361 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     3421 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     3481 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     3541 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     3601 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     3661 gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac
     3721 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc
     3781 ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag
     3841 gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg
     3901 cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag
     3961 gccattatcg ccggcatggc ggccgacgcg ctgggctacg tcttgctggc gttcgcgacg
     4021 cgaggctgga tggccttccc cattatgatt cttctcgctt ccggcggcat cgggatgccc
     4081 gcgttgcagg ccatgctgtc caggcaggta gatgacgacc atcagggaca gcttcaagga
     4141 tcgctcgcgg ctcttaccag cctaacttcg atcattggac cgctgatcgt cacggcgatt
     4201 tatgccgcct cggcgagcac atggaacggg ttggcatgga ttgtaggcgc cgccctatac
     4261 cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg
     4321 gaagccggcg gcacctcgct aacggattca ccactccaag aattggagcc aatcaattct
     4381 tgcggagaac tgtgaatgcg caaaccaacc cttggcagaa catatccatc gcgtccgcca
     4441 tctccagcag ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca
     4501 tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc
     4561 agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga
     4621 cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga
     4681 agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct
     4741 gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct
     4801 ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat
     4861 gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc
     4921 ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc
     4981 ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc
     5041 tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt
     5101 accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     5161 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     5221 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
     5281 gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccattg
     5341 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
     5401 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5461 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5521 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5581 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5641 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5701 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5761 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5821 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5881 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5941 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6001 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6061 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6121 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6181 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6241 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6301 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6361 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6421 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagagcca
     6481 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6541 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6601 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg
     6661 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6721 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6781 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6841 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6901 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat
     6961 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7021 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7081 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7141 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7201 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7261 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7321 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     7381 aggccctttc gtcttcaaga a
//
