LOCUS       sequence-298689-        7458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of biosynthetic enzyme 4CL in fusion with CC segment P3S.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7458)
  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 7458)
  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
FEATURES             Location/Qualifiers
     source          1..7458
                     /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(2080..2094)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(2110..2139)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             2152..2174
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2189..2213
                     /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(2213..2232)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2214..2232)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2316..2335)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2541..2618
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2619..3701
                     /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(2638..2657)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5013..5204
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5198..5220)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5306..5445
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5361..5380
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5460..5477)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5631..6219)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5711..5730)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6390..7181,7182..7250))
                     /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     7013..7032
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7251..7355)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7423..7441
                     /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 atccttactg ctgttcgaac tggaactgga atttctgctg
      361 ggagccgccg gagccggagc cggagccccc acccagacca tttgccagtt ttgcacgcag
      421 atctttacgc agaattttac cgctcggtgc tttcggaatg ctctcggtaa aaaacacttt
      481 gttgatgcgt ttgtagaaca ccacctgttt gctaacaaac tgtttaacat catcttcgct
      541 cagttcgcta tctttgcttt tcacaacaaa tgcaaccgga acttcgcctg ctgcttcttc
      601 tttcattgca acaactgcaa catcggtaat atccggatga ccaatcagca gtgcttccag
      661 ttctgccggt gcaacctgaa aacctttgta tttgatcagc tctttcagac gatccacaat
      721 aaacagttcg tcatcatcat caatcagacc aatatcacct gtatgcagcc aaccatcttt
      781 atcaatggtt tctgcggttg ctgccggatt attcagatag cctttcataa tctggtgacc
      841 acgaatacaa atttcaccag gctgattacg gctcaggcta tcaccggtat ccggatcaac
      901 aattttcatt tctgcattac gaacaacggt gccacatgca ccacttttaa ccggaaacgg
      961 ttcttttgca aaacccaggc tcattgccag aaccggacct gcttcggtca taccataacc
     1021 ctgacccagt tttgcattcg gaaatttggc attaactgca tcttccagtt ctttacccag
     1081 cggagctgca ccgcttttaa caacacgaat gctgctcaga tcatattttt cggtttcgct
     1141 gctttttgca attgccagaa caataggcgg aaccatcggt gcaacggtaa ctttacaacg
     1201 ctggatcagt tccagcagca gattaatttc aaatttcggc ataatcagaa ttgctgcacc
     1261 aacacgcaga ccacacagca taatgctatt cagtgcataa atatgaaaca tcggcagcac
     1321 acacagaata acatcgtcgc tatgaaaata cagattcgga ttttcaccat caacctgctg
     1381 tgcaacgctg gtaaccagac ctttatgggt cagcataaca cctttcggca gaccggtggt
     1441 gccgctgcta tacggcagtg caacaacatc atccggacta atttcaacgc tatcaataac
     1501 ttcgcttgct tcggtggtgc tctgggtcag ttcggtaaaa cgcagacaac cttccggaat
     1561 cggaacgctt tcattatcat cgatgcaaac aatcacaaca ccatcatcat tttgcagcgg
     1621 tttgattttg tccacgtaac gtgcttcggt aataatcagt ttggtgttgc ttgcttttgc
     1681 ctgttttgca atttctgccg gtgtaaaaaa cggatttgct gcggttgcgg ttgcaccacg
     1741 aaagctggct gccagaaagc tcagaacaaa ttccggacaa ttcggcagca gcagcataac
     1801 aacgtcattc tgattcacac ccagtttatg aaaattggct gcaatctgac ggctgatcac
     1861 atgaacatca ctataggtat aaacatgacc ggtcggacca ttaatcagac acggtttggt
     1921 ggcaaactcg ctaatgttct gaaagatata atcgtgcagg ctcagatgat tcggaatata
     1981 gatatccggc agtttgctac gaaaaatcac gtcgctgttg ttattgttgc tctgtttttc
     2041 cataacctgg ctaactgcct gttcttgcgg tgccatatgc ttgtcgtcgt cgtcgatatg
     2101 gccgctgctg tgatgatgat gatgatgatg atgatgatgg cccatggtat atctccttct
     2161 taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag
     2221 tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc tacgccggac gcatcgtggc
     2281 cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg
     2341 ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc
     2401 aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat tccttgcggc
     2461 ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa
     2521 gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc ggtatggcat
     2581 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
     2641 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     2701 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     2761 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     2821 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     2881 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     2941 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     3001 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     3061 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
     3121 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     3181 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
     3241 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
     3301 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
     3361 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
     3421 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca
     3481 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
     3541 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
     3601 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     3661 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta
     3721 agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca
     3781 gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc
     3841 tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt cggcgaggac
     3901 cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac
     3961 gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga gaagcaggcc
     4021 attatcgccg gcatggcggc cgacgcgctg ggctacgtct tgctggcgtt cgcgacgcga
     4081 ggctggatgg ccttccccat tatgattctt ctcgcttccg gcggcatcgg gatgcccgcg
     4141 ttgcaggcca tgctgtccag gcaggtagat gacgaccatc agggacagct tcaaggatcg
     4201 ctcgcggctc ttaccagcct aacttcgatc attggaccgc tgatcgtcac ggcgatttat
     4261 gccgcctcgg cgagcacatg gaacgggttg gcatggattg taggcgccgc cctatacctt
     4321 gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac ctgaatggaa
     4381 gccggcggca cctcgctaac ggattcacca ctccaagaat tggagccaat caattcttgc
     4441 ggagaactgt gaatgcgcaa accaaccctt ggcagaacat atccatcgcg tccgccatct
     4501 ccagcagccg cacgcggcgc atctcgggca gcgttgggtc ctggccacgg gtgcgcatga
     4561 tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga
     4621 atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct
     4681 gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt
     4741 cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg
     4801 gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt
     4861 cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt
     4921 catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca
     4981 tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta
     5041 acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg
     5101 acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc
     5161 gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     5221 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     5281 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     5341 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accattgcgg
     5401 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc
     5461 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5521 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5581 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5641 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5701 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5761 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5821 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5881 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5941 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6001 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6061 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6121 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6181 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6241 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6301 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6361 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6421 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6481 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6541 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6601 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6661 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg
     6721 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6781 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6841 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6901 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6961 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc
     7021 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7081 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7141 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7201 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7261 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7321 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7381 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     7441 ccctttcgtc ttcaagaa
//
