LOCUS       sequence-329489-        7133 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 7133)
  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 7133)
  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(N180)
FEATURES             Location/Qualifiers
     source          1..7133
                     /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             966..1217
                     /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     1154..1175
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1281..1302)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     terminator      1959..2006
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2181..2186,2187..2203,2204..2209))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2236..2254)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2322..2426
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2427..2495,2496..3287)
                     /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(2645..2664)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3458..4046
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3947..3966
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4200..4217
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4232..4371
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4297..4316)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4457..4479
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4473..4664)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5976..7058)
                     /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     7020..7039
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(7059..7133,1..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 gggtcaaact ttactggacg
      421 ccttgaatgt gagggttgta ggcagcggag agagagtgtt ggtacttgct cacgggtttg
      481 gtaccgacca gtccgcttgg aataggattc tacccttttt ccttagggac tatagagttg
      541 tactatacga tttggtgtgt gcaggatcag ttaaccctga cttctttgat tttaggaggt
      601 acacaacact tgacccttat gtggatgatt tgctgcatat attagacgcg ctggggatag
      661 actgttgcgc ttatgttggt cattcagtat cagcaatgat cggcatattg gcgtctatac
      721 gtaggccgga attattttct aagctaattt tgataggcgc gagtcctagg tttctaaacg
      781 atgaagacta ccacggcggt ttcgagcaag gagaaatcga aaaggtcttt tccgcgatgg
      841 aggcaaacta tgaggcgtgg gtgaatggat tcgcgccttt ggcagtgggc gctgatgtac
      901 cggcagcggt aagggaattc tcccgtacac tgtttaacgc atcgtataac gtctttatca
      961 tggccgacaa gcagaagaac ggcatcaagg cgaacttcaa gatccgccac aacatcgagg
     1021 acggcggcgt gcagctcgcc tatcactacc agcagaacac ccccatcggc gacggccccg
     1081 tgctgctgcc cgacaaccac tacctgagcg tgcagtccaa actgagcaaa gaccccaacg
     1141 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     1201 tggacgagct gtacaagggc ggtaccggag ggagcatggt gagcaagggc gaggagctgt
     1261 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1321 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     1381 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     1441 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1501 tgcccgaagg ctacattcag gagcgcacca tcttcttcaa ggacgacggc aactataaga
     1561 cacgcgctga ggttaagttc gagggcgaca ctctggttaa ccgcatcgag ctgaagggca
     1621 tcgacttcaa ggaggacggc aacatcctgg gccataagct tgaatataac ttcaacgcgt
     1681 ccatgcgtcc agacataact ttgttcgtta gtaggacagt attcaacagc gatatgagag
     1741 gtgttttggg cttagtgaag gtcccttgtc atatctttca gacagctaga gatcattctg
     1801 tgcctgcgag tgtagccact tatctaaaga accacctagg cggtaaaaat acagtgcact
     1861 ggcttaacat agaaggtcac cttccacatc taagtgcgcc cactctttta gcacaagagc
     1921 ttaggcgtgc actgtctcat cgttaagctg agcaataact agcataaccc cttggggcct
     1981 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga tatcccgcaa
     2041 gaggcccggc agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc
     2101 gaggatgacg atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt
     2161 taactgtgat aaactaccgc attaaagctt atcgatgata agctgtcaaa catgagaatt
     2221 cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     2281 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2341 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2401 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     2461 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     2521 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2581 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     2641 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
     2701 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     2761 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     2821 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     2881 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     2941 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     3001 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3061 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3121 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     3181 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     3241 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     3301 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     3361 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3421 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3481 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3541 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3601 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3661 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3721 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3781 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3841 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3901 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3961 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4021 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4081 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4141 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4201 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     4261 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     4321 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     4381 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4441 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4501 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     4561 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     4621 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     4681 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     4741 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     4801 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     4861 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     4921 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     4981 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     5041 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     5101 aacgacagga gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc
     5161 cgcgtgcggc tgctggagat ggcggacgcg atggatatgt tctgccaagg gttggtttgc
     5221 gcattcacag ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta
     5281 gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca
     5341 acgcggggag gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg
     5401 tgctcgccga ggcggcataa atcgccgtga cgatcagcgg tccaatgatc gaagttaggc
     5461 tggtaagagc cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg
     5521 acagcatggc ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg
     5581 ggaaggccat ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg
     5641 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     5701 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     5761 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     5821 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     5881 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     5941 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     6001 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6061 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     6121 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     6181 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     6241 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     6301 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     6361 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     6421 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     6481 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     6541 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     6601 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     6661 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     6721 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     6781 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     6841 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     6901 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     6961 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     7021 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     7081 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggt
//
