LOCUS       sequence-308958-        8852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of biosynthetic enzymes 4CL and STS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8852)
  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 8852)
  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
FEATURES             Location/Qualifiers
     source          1..8852
                     /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(1327..1341)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(1357..1386)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1399..1421
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1436..1460
                     /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(1461..1479)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(1664..1711)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3474..3488)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(3504..3533)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             3546..3568
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3583..3607
                     /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(3608..3626)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3710..3729)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3935..4012
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4013..5095
                     /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(4032..4051)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6407..6598
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6592..6614)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6700..6839
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6755..6774
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6854..6871)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7025..7613)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7105..7124)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7784..8575,8576..8644))
                     /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     8407..8426
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8645..8749)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8817..8835
                     /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 aattggtaac catcggaatg ctatgcagaa caacggtttc
      181 aatggtcaga cccggaccaa aaccaaacag aacaccccaa tccagacctt caccggtggt
      241 tgcacgttca cctttcaggc tttttttgcg catttcatcc agaataaaca gaacacatgc
      301 gctgctcata ttaccatatt cgctcagaac atgacgggtt gcttccagtt tctttttgtc
      361 caggttcagt tttgcttcaa ctgcatccag aattgccgga ccacccggat gtgcaatcca
      421 aaacaggcta ttccaatcgc taatacccag cggatcaaat gcctgggtca gacatttttc
      481 aacattttcg ctaatcaggg tcggaacatt cggccacaga tgaaaggtca gaccaacttc
      541 acgcagatta cctgcaattg caccggcact attcggaata aaggtctgtg ctgcgctaac
      601 cagctgaaac agcggacgtt caatgctaat atccggatcg ctaccaacaa taactgctgc
      661 gctaccatca ccaaacagtg cctgaccaac caggctatcc agtgcatctt cgctcggacc
      721 acgaaaggta acaacggtaa tttcgctaca aacaaccaga acacgtgcac cggcattatt
      781 ttctgccaga tcttttgcgg tacgcagaac ggtgccaccg gcataacaac cctgatgata
      841 cagcataaca cgacgaacgc tcggttccag acccagcaga tttgccagtt tataatctgc
      901 accaggcatt tcaacaccac tggtggtaca aaaaaccaga tgggtaattt tgcttttcgg
      961 ctgaccccat tctttcaggg ctttcagtgc tgcttcttta cccagtttcg gaacttctgc
     1021 ggtgataatt tcttgacgaa tattcaggct cggtgccata tatgcaccaa tattcggatg
     1081 ttcttccagc atttcttcgg tcaggtggat atagcgtttt ttgatcatgc ttttatcgca
     1141 gatgcggtta aatttctttt tcagtgcggt catgtgttcg cttttggtca ctttaaagta
     1201 ataatcggca taatcgctct gataaacaca atgatccggt gttgcggtgc caattgccag
     1261 aatggttgcc ggaccttttg cacgctgtgc attacgaaat tcttcaacgc ttgcctcgag
     1321 catatgcttg tcgtcgtcgt cgatatggcc gctgctgtga tgatgatgat gatgatgatg
     1381 atgatggccc atggtatatc tccttcttaa agttaaacaa aattatttct agaggggaat
     1441 tgttatccgc tcacaattcc cctatagtga gtcgtattaa agctttaatg cggtagttta
     1501 tcacagttaa attgctaacg cagtcaggca ccgtgtatga aatctaacaa tgcgctcatc
     1561 gtcatcctcg gcaccgtcac cctggatgct gtaggcatag gcttggttat gccggtactg
     1621 ccgggcctct tgcgggatat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga
     1681 cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact
     1741 cagcttcctt tcgggctttg ttagcagccg gatccttaac ccagaccatt tgccagtttt
     1801 gcacgcagat ctttacgcag aattttaccg ctcggtgctt tcggaatgct ctcggtaaaa
     1861 aacactttgt tgatgcgttt gtagaacacc acctgtttgc taacaaactg tttaacatca
     1921 tcttcgctca gttcgctatc tttgcttttc acaacaaatg caaccggaac ttcgcctgct
     1981 gcttcttctt tcattgcaac aactgcaaca tcggtaatat ccggatgacc aatcagcagt
     2041 gcttccagtt ctgccggtgc aacctgaaaa cctttgtatt tgatcagctc tttcagacga
     2101 tccacaataa acagttcgtc atcatcatca atcagaccaa tatcacctgt atgcagccaa
     2161 ccatctttat caatggtttc tgcggttgct gccggattat tcagatagcc tttcataatc
     2221 tggtgaccac gaatacaaat ttcaccaggc tgattacggc tcaggctatc accggtatcc
     2281 ggatcaacaa ttttcatttc tgcattacga acaacggtgc cacatgcacc acttttaacc
     2341 ggaaacggtt cttttgcaaa acccaggctc attgccagaa ccggacctgc ttcggtcata
     2401 ccataaccct gacccagttt tgcattcgga aatttggcat taactgcatc ttccagttct
     2461 ttacccagcg gagctgcacc gcttttaaca acacgaatgc tgctcagatc atatttttcg
     2521 gtttcgctgc tttttgcaat tgccagaaca ataggcggaa ccatcggtgc aacggtaact
     2581 ttacaacgct ggatcagttc cagcagcaga ttaatttcaa atttcggcat aatcagaatt
     2641 gctgcaccaa cacgcagacc acacagcata atgctattca gtgcataaat atgaaacatc
     2701 ggcagcacac acagaataac atcgtcgcta tgaaaataca gattcggatt ttcaccatca
     2761 acctgctgtg caacgctggt aaccagacct ttatgggtca gcataacacc tttcggcaga
     2821 ccggtggtgc cgctgctata cggcagtgca acaacatcat ccggactaat ttcaacgcta
     2881 tcaataactt cgcttgcttc ggtggtgctc tgggtcagtt cggtaaaacg cagacaacct
     2941 tccggaatcg gaacgctttc attatcatcg atgcaaacaa tcacaacacc atcatcattt
     3001 tgcagcggtt tgattttgtc cacgtaacgt gcttcggtaa taatcagttt ggtgttgctt
     3061 gcttttgcct gttttgcaat ttctgccggt gtaaaaaacg gatttgctgc ggttgcggtt
     3121 gcaccacgaa agctggctgc cagaaagctc agaacaaatt ccggacaatt cggcagcagc
     3181 agcataacaa cgtcattctg attcacaccc agtttatgaa aattggctgc aatctgacgg
     3241 ctgatcacat gaacatcact ataggtataa acatgaccgg tcggaccatt aatcagacac
     3301 ggtttggtgg caaactcgct aatgttctga aagatataat cgtgcaggct cagatgattc
     3361 ggaatataga tatccggcag tttgctacga aaaatcacgt cgctgttgtt attgttgctc
     3421 tgtttttcca taacctggct aactgcctgt tcttgcggtg cctcgagcat atgcttgtcg
     3481 tcgtcgtcga tatggccgct gctgtgatga tgatgatgat gatgatgatg atggcccatg
     3541 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca
     3601 caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc
     3661 ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc
     3721 gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc
     3781 gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca
     3841 ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg
     3901 caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt
     3961 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     4021 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     4081 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     4141 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     4201 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     4261 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     4321 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     4381 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     4441 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     4501 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     4561 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     4621 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     4681 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     4741 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     4801 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     4861 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     4921 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     4981 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     5041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     5101 acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga
     5161 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     5221 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca
     5281 ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat
     5341 tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg
     5401 gcgagaagca ggccattatc gccggcatgg cggccgacgc gctgggctac gtcttgctgg
     5461 cgttcgcgac gcgaggctgg atggccttcc ccattatgat tcttctcgct tccggcggca
     5521 tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt agatgacgac catcagggac
     5581 agcttcaagg atcgctcgcg gctcttacca gcctaacttc gatcattgga ccgctgatcg
     5641 tcacggcgat ttatgccgcc tcggcgagca catggaacgg gttggcatgg attgtaggcg
     5701 ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc cgggccacct
     5761 cgacctgaat ggaagccggc ggcacctcgc taacggattc accactccaa gaattggagc
     5821 caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga acatatccat
     5881 cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg ggcagcgttg ggtcctggcc
     5941 acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct
     6001 tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa
     6061 aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct
     6121 ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg
     6181 ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt
     6241 gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag
     6301 taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg
     6361 tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga
     6421 aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa
     6481 actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc
     6541 tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     6601 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     6661 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     6721 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     6781 gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     6841 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     6901 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     6961 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     7021 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     7081 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     7141 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     7201 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     7261 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     7321 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     7381 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     7441 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     7501 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     7561 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     7621 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     7681 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     7741 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     7801 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     7861 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     7921 cgcgagagcc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     7981 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     8041 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg
     8101 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     8161 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     8221 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     8281 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     8341 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     8401 cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     8461 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     8521 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     8581 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     8641 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     8701 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     8761 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     8821 ggcgtatcac gaggcccttt cgtcttcaag aa
//
