LOCUS       sequence-329416-        7889 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of coding sequence in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7889)
  AUTHORS   Chesterfield RJ, Whitfield JH, Pouvreau B, Cao D, Alexandrov K, 
            Beveridge CA, Vickers CE
  TITLE     Rational Design of Novel Fluorescent Enzyme Biosensors for Direct 
            Detection of Strigolactones.
  JOURNAL   ACS Synth Biol. 2020 Aug 21;9(8):2107-2118. doi: 
            10.1021/acssynbio.0c00192. Epub 2020 Aug 11.
  PUBMED    32786922
REFERENCE   2  (bases 1 to 7889)
  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"; doi: 
            "10.1021/acssynbio.0c00192"; journalName: "ACS Synth Biol"; date: 
            "2020-08-21- 21"; volume: "9"; issue: "8"; pages: "2107-2118"
COMMENT     Sequence Label: pET19b-rShHTL7 cPGFP(L166)
FEATURES             Location/Qualifiers
     source          1..7889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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)."
     RBS             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             594..845
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNH
                     YLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     782..803
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(909..930)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             1665..2372
                     /codon_start=1
                     /product="monomeric orange fluorescent protein with a large
                     Stokes shift (Shcherbakova et al., 2012)"
                     /label=LSSmOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALK
                     GEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     2078..2097
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      2385..2432
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2607..2612,2613..2629,2630..2635))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2662..2680)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2748..2852
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2853..2921,2922..3713)
                     /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     complement(3071..3090)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3884..4472
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4373..4392
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4626..4643
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4658..4797
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4723..4742)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4883..4905
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4899..5090)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6402..7484)
                     /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     7446..7465
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7485..7562)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7768..7787
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(7871..7889,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7871..7889
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gagtagcatt gggttagcgc acaatgttac gatcctggga agcggagaaa
      121 ctaccgtggt attgggtcac ggatacggca ctgatcagtc tgtgtggaaa ctgcttgtac
      181 cgtacttggt tgatgattac aaggtcttac tttacgatca tatgggggcg ggcacaacca
      241 accctgatta tttcgatttc gaccgctata gttcactgga gggctacagt tacgacttga
      301 tcgcaatttt ggaagagttc caagtatcga agtgcattta cgttggacac tcgatgtctt
      361 cgatggcagc ggcagtcgca tctatttttc gccctgatct gtttcacaaa ttagtaatga
      421 tcagccccac accccgcctt attaacactg aagaatacta cggtggattt gagcaaaagg
      481 ttatggacga gaccctgcgc tcgttagacg aaaactttaa aagcctgtcg ttgggtacgg
      541 caccgttgtt gttggcttgt gatttggcat cgtataacgt ctttatcatg gccgacaagc
      601 agaagaacgg catcaaggcg aacttcaaga tccgccacaa catcgaggac ggcggcgtgc
      661 agctcgccta tcactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
      721 acaaccacta cctgagcgtg cagtccaaac tgagcaaaga ccccaacgag aagcgcgatc
      781 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
      841 acaagggcgg taccggaggg agcatggtga gcaagggcga ggagctgttc accggggtgg
      901 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
      961 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1021 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     1081 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1141 acattcagga gcgcaccatc ttcttcaagg acgacggcaa ctataagaca cgcgctgagg
     1201 ttaagttcga gggcgacact ctggttaacc gcatcgagct gaagggcatc gacttcaagg
     1261 aggacggcaa catcctgggc cataagcttg aatataactt caacgcgtcc gagagtgcag
     1321 ccatgcagga gtactgccgt actctgttca acatgcgccc ggacattgct tgctgcatta
     1381 cgcgtatgat ttgcgggctg gatttgcgtc catatctggg acacgtgacc gttccatgcc
     1441 acatcattca aagctcgaac gacatcatgg ttccggtcgc ggtgggggag tacttgcgta
     1501 agaatcttgg tggcccatcg gtcgttgagg tgatgcccac cgagggacat ttaccgcatt
     1561 tgagtatgcc tgaagttact atccctgtag tgttacgtca cattcgccaa gacatcacgg
     1621 accacggaga agcagcagca aaggaagcgg cggcaaaagg gggcatggtg agcaagggcg
     1681 aggagaataa catggccatc atcaaggagt tcatgcgctt caaggtgcgc atggagggct
     1741 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggct
     1801 ttcagaccgt taagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatct
     1861 tgtcccctca gttcacctac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     1921 actacctcaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     1981 acggcggcgt ggtgaccgtg actcaggact cctccctgca ggacggcgag ttcatctaca
     2041 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
     2101 tgggcatgga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgagg
     2161 acaagctcag gctgaagctg aaggacggcg gccactacac ctccgaggtc aagaccacct
     2221 acaaggccaa gaagcccgtg cagttgcccg gcgcctacat cgtcgacatc aagttggaca
     2281 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     2341 actccaccgg cggcatggac gagctgtaca aggctgagca ataactagca taaccccttg
     2401 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc
     2461 ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac
     2521 ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta
     2581 gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg
     2641 agaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     2701 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2761 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2821 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     2881 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2941 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     3001 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     3061 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
     3121 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     3181 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     3241 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     3301 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3361 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     3421 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3481 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3541 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     3601 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3661 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3721 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3781 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3841 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3901 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3961 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4021 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4081 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4141 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4201 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4261 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4321 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4381 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4441 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4501 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4561 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4621 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4681 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     4741 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     4801 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4861 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4921 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     4981 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     5041 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     5101 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     5161 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     5221 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     5281 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     5341 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     5401 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     5461 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     5521 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag
     5581 atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg
     5641 gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat
     5701 ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc
     5761 gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt
     5821 tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca atgatcgaag
     5881 ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct
     5941 gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca
     6001 taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt
     6061 cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     6121 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     6181 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     6241 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     6301 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     6361 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     6421 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6481 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     6541 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     6601 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     6661 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     6721 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     6781 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     6841 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     6901 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     6961 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     7021 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     7081 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     7141 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     7201 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     7261 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     7321 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     7381 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     7441 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     7501 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     7561 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     7621 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     7681 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     7741 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     7801 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     7861 cccgcgaaat taatacgact cactatagg
//
