LOCUS       sequence-329439-        7172 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 7172)
  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 7172)
  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-HisDAD2 cPGFP(G164)
FEATURES             Location/Qualifiers
     source          1..7172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     209..228
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     312..331
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        312..330
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    331..355
                     /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             370..392
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             405..422
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             423..443
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             957..1208
                     /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     1145..1166
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1272..1293)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     terminator      1998..2045
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2220..2225,2226..2242,2243..2248))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2275..2293)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2361..2465
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2466..2534,2535..3326)
                     /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(2684..2703)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3497..4085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3986..4005
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4239..4256
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4271..4410
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4336..4355)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4496..4518
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4512..4703)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6015..7097)
                     /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     7059..7078
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(7098..7172,1..3))
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
       61 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
      121 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
      181 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
      241 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
      301 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
      361 agaaataatt ttgtttaact ttaagaagga gatataccat gggtcatcac catcaccatc
      421 acgagaacct gtattttcag ggtcaaactt tactggacgc cttgaatgtg agggttgtag
      481 gcagcggaga gagagtgttg gtacttgctc acgggtttgg taccgaccag tccgcttgga
      541 ataggattct accctttttc cttagggact atagagttgt actatacgat ttggtgtgtg
      601 caggatcagt taaccctgac ttctttgatt ttaggaggta cacaacactt gacccttatg
      661 tggatgattt gctgcatata ttagacgcgc tggggataga ctgttgcgct tatgttggtc
      721 attcagtatc agcaatgatc ggcatattgg cgtctatacg taggccggaa ttattttcta
      781 agctaatttt gataggcgcg agtcctaggt ttctaaacga tgaagactac cacggcggtt
      841 tcgagcaagg agaaatcgaa aaggtctttt ccgcgatgga ggcaaactat gaggcgtggg
      901 tgaatggatt cgcgcctttg gcagtgggcg catcgtataa cgtctttatc atggccgaca
      961 agcagaagaa cggcatcaag gcgaacttca agatccgcca caacatcgag gacggcggcg
     1021 tgcagctcgc ctatcactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1081 ccgacaacca ctacctgagc gtgcagtcca aactgagcaa agaccccaac gagaagcgcg
     1141 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     1201 tgtacaaggg cggtaccgga gggagcatgg tgagcaaggg cgaggagctg ttcaccgggg
     1261 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1321 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1381 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1441 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1501 gctacattca ggagcgcacc atcttcttca aggacgacgg caactataag acacgcgctg
     1561 aggttaagtt cgagggcgac actctggtta accgcatcga gctgaagggc atcgacttca
     1621 aggaggacgg caacatcctg ggccataagc ttgaatataa cttcaacgcg tccgctgatg
     1681 taccggcagc ggtaagggaa ttctcccgta cactgtttaa catgcgtcca gacataactt
     1741 tgttcgttag taggacagta ttcaacagcg atatgagagg tgttttgggc ttagtgaagg
     1801 tcccttgtca tatctttcag acagctagag atcattctgt gcctgcgagt gtagccactt
     1861 atctaaagaa ccacctaggc ggtaaaaata cagtgcactg gcttaacata gaaggtcacc
     1921 ttccacatct aagtgcgccc actcttttag cacaagagct taggcgtgca ctgtctcatc
     1981 gttaagctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     2041 ttttgctgaa aggaggaact atatccggat atcccgcaag aggcccggca gtaccggcat
     2101 aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt
     2161 gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca
     2221 ttaaagctta tcgatgataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg
     2281 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     2341 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2401 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2461 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     2521 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     2581 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     2641 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     2701 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2761 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2821 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     2881 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     2941 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     3001 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3061 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3121 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     3181 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     3241 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     3301 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     3361 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     3421 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3481 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3541 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3601 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3661 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3721 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3781 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3841 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3901 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3961 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4021 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4081 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4141 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4201 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4261 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4321 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     4381 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     4441 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4501 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     4561 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     4621 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     4681 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     4741 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     4801 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     4861 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     4921 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     4981 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     5041 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     5101 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     5161 cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg
     5221 gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag
     5281 aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca
     5341 ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt
     5401 atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa
     5461 tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc
     5521 cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg
     5581 gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg
     5641 tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct
     5701 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     5761 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     5821 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     5881 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     5941 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     6001 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     6061 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     6121 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     6181 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     6241 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     6301 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     6361 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     6421 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     6481 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     6541 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     6601 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     6661 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     6721 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     6781 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     6841 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     6901 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     6961 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     7021 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     7081 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     7141 ataccgcgaa aggttttgcg ccattcgatg gt
//
