LOCUS       sequence-329459-        7877 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 7877)
  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 7877)
  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-DAD2 cPGFP(A149)
FEATURES             Location/Qualifiers
     source          1..7877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     186..207
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(313..334)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             1108..1815
                     /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     1521..1540
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      1828..1875
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2050..2055,2056..2072,2073..2078))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2105..2123)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2191..2295
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2296..2364,2365..3156)
                     /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(2514..2533)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3327..3915
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3816..3835
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4069..4086
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4101..4240
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4166..4185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4326..4348
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4342..4533)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5845..6927)
                     /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     6889..6908
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6928..7005)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7211..7230
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7314..7333
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7314..7332
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7333..7357
                     /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             7372..7394
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(7875..7877,1..249)
                     /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"
ORIGIN
        1 aagcagaaga acggcatcaa ggcgaacttc aagatccgcc acaacatcga ggacggcggc
       61 gtgcagctcg cctatcacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
      121 cccgacaacc actacctgag cgtgcagtcc aaactgagca aagaccccaa cgagaagcgc
      181 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
      241 ctgtacaagg gcggtaccgg agggagcatg gtgagcaagg gcgaggagct gttcaccggg
      301 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
      361 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
      421 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
      481 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
      541 ggctacattc aggagcgcac catcttcttc aaggacgacg gcaactataa gacacgcgct
      601 gaggttaagt tcgagggcga cactctggtt aaccgcatcg agctgaaggg catcgacttc
      661 aaggaggacg gcaacatcct gggccataag cttgaatata acttcaacgc gtccaactat
      721 gaggcgtggg tgaatggatt cgcgcctttg gcagtgggcg ctgatgtacc ggcagcggta
      781 agggaattct cccgtacact gtttaacatg cgtccagaca taactttgtt cgttagtagg
      841 acagtattca acagcgatat gagaggtgtt ttgggcttag tgaaggtccc ttgtcatatc
      901 tttcagacag ctagagatca ttctgtgcct gcgagtgtag ccacttatct aaagaaccac
      961 ctaggcggta aaaatacagt gcactggctt aacatagaag gtcaccttcc acatctaagt
     1021 gcgcccactc ttttagcaca agagcttagg cgtgcactgt ctcatcgtgg agaagcagca
     1081 gcaaaggaag cggcggcaaa agggggcatg gtgagcaagg gcgaggagaa taacatggcc
     1141 atcatcaagg agttcatgcg cttcaaggtg cgcatggagg gctccgtgaa cggccacgag
     1201 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gctttcagac cgttaagctg
     1261 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcttgtcccc tcagttcacc
     1321 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactacct caagctgtcc
     1381 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     1441 gtgactcagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     1501 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggcat ggaggcctcc
     1561 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg aggacaagct caggctgaag
     1621 ctgaaggacg gcggccacta cacctccgag gtcaagacca cctacaaggc caagaagccc
     1681 gtgcagttgc ccggcgccta catcgtcgac atcaagttgg acatcacctc ccacaacgag
     1741 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     1801 gacgagctgt acaaggctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1861 ttgaggggtt ttttgctgaa aggaggaact atatccggat atcccgcaag aggcccggca
     1921 gtaccggcat aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga
     1981 tgagcgcatt gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata
     2041 aactaccgca ttaaagctta tcgatgataa gctgtcaaac atgagaattc ttgaagacga
     2101 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     2161 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2221 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2281 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2341 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2401 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2461 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2521 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     2581 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2641 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2701 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2761 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2821 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2881 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2941 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3001 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3061 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3121 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3181 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3241 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3301 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3361 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3421 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3481 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3541 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3601 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3661 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3721 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3781 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3841 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3901 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3961 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4021 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4081 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4141 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4201 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4261 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4321 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4381 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     4441 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     4501 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     4561 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     4621 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     4681 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     4741 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4801 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     4861 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     4921 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     4981 cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct
     5041 gctggagatg gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt
     5101 tctccgcaag aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg
     5161 ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg
     5221 cagacaaggt atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag
     5281 gcggcataaa tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc
     5341 gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc
     5401 tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc
     5461 cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg
     5521 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     5581 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     5641 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     5701 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     5761 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     5821 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5881 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     5941 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     6001 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     6061 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     6121 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     6181 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     6241 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     6301 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     6361 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     6421 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     6481 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     6541 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     6601 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     6661 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     6721 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     6781 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     6841 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     6901 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     6961 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     7021 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     7081 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     7141 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     7201 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     7261 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     7321 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     7381 ctttaagaag gagatatacc atgggtcaaa ctttactgga cgccttgaat gtgagggttg
     7441 taggcagcgg agagagagtg ttggtacttg ctcacgggtt tggtaccgac cagtccgctt
     7501 ggaataggat tctacccttt ttccttaggg actatagagt tgtactatac gatttggtgt
     7561 gtgcaggatc agttaaccct gacttctttg attttaggag gtacacaaca cttgaccctt
     7621 atgtggatga tttgctgcat atattagacg cgctggggat agactgttgc gcttatgttg
     7681 gtcattcagt atcagcaatg atcggcatat tggcgtctat acgtaggccg gaattatttt
     7741 ctaagctaat tttgataggc gcgagtccta ggtttctaaa cgatgaagac taccacggcg
     7801 gtttcgagca aggagaaatc gaaaaggtct tttccgcgat ggaggcagca tcgtataacg
     7861 tctttatcat ggccgac
//
