LOCUS       sequence-298687-        9056 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 9056)
  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 9056)
  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:P7S + STS:P8S
FEATURES             Location/Qualifiers
     source          1..9056
                     /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(1429..1443)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(1459..1488)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1501..1523
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1538..1562
                     /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(1563..1581)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(1766..1813)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3678..3692)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(3708..3737)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             3750..3772
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3787..3811
                     /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(3812..3830)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3914..3933)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4139..4216
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4217..5299
                     /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(4236..4255)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6611..6802
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6796..6818)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6904..7043
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6959..6978
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(7058..7075)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7229..7817)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7309..7328)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7988..8779,8780..8848))
                     /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     8611..8630
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8849..8953)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     9021..9039
                     /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 acccgtactc cagttgtgag ttttcttcct ttaactgttg
      181 gattttctgc tctaactgtt gattttcctc tttaagctgt gaaatcttgt cctctggtga
      241 gccgctgcca ttggtaacca tcggaatgct atgcagaaca acggtttcaa tggtcagacc
      301 cggaccaaaa ccaaacagaa caccccaatc cagaccttca ccggtggttg cacgttcacc
      361 tttcaggctt tttttgcgca tttcatccag aataaacaga acacatgcgc tgctcatatt
      421 accatattcg ctcagaacat gacgggttgc ttccagtttc tttttgtcca ggttcagttt
      481 tgcttcaact gcatccagaa ttgccggacc acccggatgt gcaatccaaa acaggctatt
      541 ccaatcgcta atacccagcg gatcaaatgc ctgggtcaga catttttcaa cattttcgct
      601 aatcagggtc ggaacattcg gccacagatg aaaggtcaga ccaacttcac gcagattacc
      661 tgcaattgca ccggcactat tcggaataaa ggtctgtgct gcgctaacca gctgaaacag
      721 cggacgttca atgctaatat ccggatcgct accaacaata actgctgcgc taccatcacc
      781 aaacagtgcc tgaccaacca ggctatccag tgcatcttcg ctcggaccac gaaaggtaac
      841 aacggtaatt tcgctacaaa caaccagaac acgtgcaccg gcattatttt ctgccagatc
      901 ttttgcggta cgcagaacgg tgccaccggc ataacaaccc tgatgataca gcataacacg
      961 acgaacgctc ggttccagac ccagcagatt tgccagttta taatctgcac caggcatttc
     1021 aacaccactg gtggtacaaa aaaccagatg ggtaattttg cttttcggct gaccccattc
     1081 tttcagggct ttcagtgctg cttctttacc cagtttcgga acttctgcgg tgataatttc
     1141 ttgacgaata ttcaggctcg gtgccatata tgcaccaata ttcggatgtt cttccagcat
     1201 ttcttcggtc aggtggatat agcgtttttt gatcatgctt ttatcgcaga tgcggttaaa
     1261 tttctttttc agtgcggtca tgtgttcgct tttggtcact ttaaagtaat aatcggcata
     1321 atcgctctga taaacacaat gatccggtgt tgcggtgcca attgccagaa tggttgccgg
     1381 accttttgca cgctgtgcat tacgaaattc ttcaacgctt gccatatgct tgtcgtcgtc
     1441 gtcgatatgg ccgctgctgt gatgatgatg atgatgatga tgatgatggc ccatggtata
     1501 tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt
     1561 cccctatagt gagtcgtatt aaagctttaa tgcggtagtt tatcacagtt aaattgctaa
     1621 cgcagtcagg caccgtgtat gaaatctaac aatgcgctca tcgtcatcct cggcaccgtc
     1681 accctggatg ctgtaggcat aggcttggtt atgccggtac tgccgggcct cttgcgggat
     1741 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
     1801 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
     1861 tgttagcagc cggatcctta accgtacttt aattgttgat tcttttcctc caactgagag
     1921 atttcctgtt taagctgact gttcttctct tccaacgact gaatctcgtc ctctggtgag
     1981 ccgctgccac ccagaccatt tgccagtttt gcacgcagat ctttacgcag aattttaccg
     2041 ctcggtgctt tcggaatgct ctcggtaaaa aacactttgt tgatgcgttt gtagaacacc
     2101 acctgtttgc taacaaactg tttaacatca tcttcgctca gttcgctatc tttgcttttc
     2161 acaacaaatg caaccggaac ttcgcctgct gcttcttctt tcattgcaac aactgcaaca
     2221 tcggtaatat ccggatgacc aatcagcagt gcttccagtt ctgccggtgc aacctgaaaa
     2281 cctttgtatt tgatcagctc tttcagacga tccacaataa acagttcgtc atcatcatca
     2341 atcagaccaa tatcacctgt atgcagccaa ccatctttat caatggtttc tgcggttgct
     2401 gccggattat tcagatagcc tttcataatc tggtgaccac gaatacaaat ttcaccaggc
     2461 tgattacggc tcaggctatc accggtatcc ggatcaacaa ttttcatttc tgcattacga
     2521 acaacggtgc cacatgcacc acttttaacc ggaaacggtt cttttgcaaa acccaggctc
     2581 attgccagaa ccggacctgc ttcggtcata ccataaccct gacccagttt tgcattcgga
     2641 aatttggcat taactgcatc ttccagttct ttacccagcg gagctgcacc gcttttaaca
     2701 acacgaatgc tgctcagatc atatttttcg gtttcgctgc tttttgcaat tgccagaaca
     2761 ataggcggaa ccatcggtgc aacggtaact ttacaacgct ggatcagttc cagcagcaga
     2821 ttaatttcaa atttcggcat aatcagaatt gctgcaccaa cacgcagacc acacagcata
     2881 atgctattca gtgcataaat atgaaacatc ggcagcacac acagaataac atcgtcgcta
     2941 tgaaaataca gattcggatt ttcaccatca acctgctgtg caacgctggt aaccagacct
     3001 ttatgggtca gcataacacc tttcggcaga ccggtggtgc cgctgctata cggcagtgca
     3061 acaacatcat ccggactaat ttcaacgcta tcaataactt cgcttgcttc ggtggtgctc
     3121 tgggtcagtt cggtaaaacg cagacaacct tccggaatcg gaacgctttc attatcatcg
     3181 atgcaaacaa tcacaacacc atcatcattt tgcagcggtt tgattttgtc cacgtaacgt
     3241 gcttcggtaa taatcagttt ggtgttgctt gcttttgcct gttttgcaat ttctgccggt
     3301 gtaaaaaacg gatttgctgc ggttgcggtt gcaccacgaa agctggctgc cagaaagctc
     3361 agaacaaatt ccggacaatt cggcagcagc agcataacaa cgtcattctg attcacaccc
     3421 agtttatgaa aattggctgc aatctgacgg ctgatcacat gaacatcact ataggtataa
     3481 acatgaccgg tcggaccatt aatcagacac ggtttggtgg caaactcgct aatgttctga
     3541 aagatataat cgtgcaggct cagatgattc ggaatataga tatccggcag tttgctacga
     3601 aaaatcacgt cgctgttgtt attgttgctc tgtttttcca taacctggct aactgcctgt
     3661 tcttgcggtg ccatatgctt gtcgtcgtcg tcgatatggc cgctgctgtg atgatgatga
     3721 tgatgatgat gatgatggcc catggtatat ctccttctta aagttaaaca aaattatttc
     3781 tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg
     3841 atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
     3901 gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc
     3961 gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg
     4021 ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac
     4081 ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga
     4141 caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     4201 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     4261 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     4321 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     4381 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     4441 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     4501 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     4561 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     4621 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     4681 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     4741 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     4801 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     4861 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     4921 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     4981 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     5041 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     5101 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     5161 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca
     5221 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5281 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     5341 cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc
     5401 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac
     5461 aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga
     5521 tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca
     5581 ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccg
     5641 acgcgctggg ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc ttccccatta
     5701 tgattcttct cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc
     5761 aggtagatga cgaccatcag ggacagcttc aaggatcgct cgcggctctt accagcctaa
     5821 cttcgatcat tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg agcacatgga
     5881 acgggttggc atggattgta ggcgccgccc tataccttgt ctgcctcccc gcgttgcgtc
     5941 gcggtgcatg gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg
     6001 attcaccact ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac
     6061 caacccttgg cagaacatat ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat
     6121 ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac
     6181 ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg
     6241 aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt
     6301 cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt
     6361 ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac
     6421 gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag
     6481 ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt
     6541 gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac
     6601 ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag
     6661 ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat
     6721 ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg
     6781 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     6841 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     6901 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg
     6961 gcatcagagc agattgtact gagagtgcac cattgcggtg tgaaataccg cacagatgcg
     7021 taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     7081 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     7141 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     7201 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     7261 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     7321 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     7381 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     7441 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     7501 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     7561 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     7621 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     7681 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     7741 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     7801 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     7861 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     7921 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     7981 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     8041 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     8101 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     8161 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     8221 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     8281 tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg
     8341 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     8401 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     8461 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     8521 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     8581 cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa
     8641 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     8701 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     8761 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     8821 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     8881 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     8941 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     9001 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaa
//
