LOCUS       sequence-329546-        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-rDAD2 cPGFP(N180)
FEATURES             Location/Qualifiers
     source          1..7877
                     /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)"
     primer_bind     824..845
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(951..972)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             1653..2360
                     /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
                     FQTAKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALK
                     GEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     2066..2085
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      2373..2420
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2595..2600,2601..2617,2618..2623))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2650..2668)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2736..2840
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2841..2909,2910..3701)
                     /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(3059..3078)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3872..4460
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4361..4380
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4614..4631
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4646..4785
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4711..4730)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4871..4893
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4887..5078)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6390..7472)
                     /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     7434..7453
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7473..7550)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7756..7775
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(7859..7877,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7859..7877
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gggtcaaact ttactggacg ccttgaatgt gagggttgta ggcagcggag
      121 agagagtgtt ggtacttgct cacgggtttg gtaccgacca gtccgcttgg aataggattc
      181 tacccttttt ccttagggac tatagagttg tactatacga tttggtgtgt gcaggatcag
      241 ttaaccctga cttctttgat tttaggaggt acacaacact tgacccttat gtggatgatt
      301 tgctgcatat attagacgcg ctggggatag actgttgcgc ttatgttggt cattcagtat
      361 cagcaatgat cggcatattg gcgtctatac gtaggccgga attattttct aagctaattt
      421 tgataggcgc gagtcctagg tttctaaacg atgaagacta ccacggcggt ttcgagcaag
      481 gagaaatcga aaaggtcttt tccgcgatgg aggcaaacta tgaggcgtgg gtgaatggat
      541 tcgcgccttt ggcagtgggc gctgatgtac cggcagcggt aagggaattc tcccgtacac
      601 tgtttaacgc atcgtataac gtctttatca tggccgacaa gcagaagaac ggcatcaagg
      661 cgaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc tatcactacc
      721 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagtg
      781 tgcagtccaa actgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
      841 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc ggtaccggag
      901 ggagcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
      961 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1021 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1081 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1141 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacattcag gagcgcacca
     1201 tcttcttcaa ggacgacggc aactataaga cacgcgctga ggttaagttc gagggcgaca
     1261 ctctggttaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1321 gccataagct tgaatataac ttcaacgcgt ccatgcgtcc agacataact ttgttcgtta
     1381 gtaggacagt attcaacagc gatatgagag gtgttttggg cttagtgaag gtcccttgtc
     1441 atatctttca gacagctaga gatcattctg tgcctgcgag tgtagccact tatctaaaga
     1501 accacctagg cggtaaaaat acagtgcact ggcttaacat agaaggtcac cttccacatc
     1561 taagtgcgcc cactctttta gcacaagagc ttaggcgtgc actgtctcat cgtggagaag
     1621 cagcagcaaa ggaagcggcg gcaaaagggg gcatggtgag caagggcgag gagaataaca
     1681 tggccatcat caaggagttc atgcgcttca aggtgcgcat ggagggctcc gtgaacggcc
     1741 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcttt cagaccgcta
     1801 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcttg tcccctcagt
     1861 tcacctacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacctcaagc
     1921 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     1981 tgaccgtgac tcaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     2041 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggcatggagg
     2101 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgaggac aagctcaggc
     2161 tgaagctgaa ggacggcggc cactacacct ccgaggtcaa gaccacctac aaggccaaga
     2221 agcccgtgca gttgcccggc gcctacatcg tcgacatcaa gttggacatc acctcccaca
     2281 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
     2341 gcatggacga gctgtacaag gctgagcaat aactagcata accccttggg gcctctaaac
     2401 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatccc gcaagaggcc
     2461 cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat
     2521 gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg
     2581 tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag aattcttgaa
     2641 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     2701 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2761 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2821 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2881 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2941 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3001 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     3061 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     3121 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     3181 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     3241 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     3301 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3361 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     3421 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     3481 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     3541 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     3601 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3661 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3721 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3781 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3841 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3901 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3961 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4021 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4081 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4141 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4201 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4261 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4321 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4381 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4441 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4501 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4561 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4621 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4681 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4741 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4801 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4861 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4921 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     4981 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     5041 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     5101 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     5161 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     5221 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     5281 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     5341 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     5401 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     5461 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     5521 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
     5581 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
     5641 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
     5701 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
     5761 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
     5821 ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa
     5881 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
     5941 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
     6001 ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc
     6061 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     6121 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     6181 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     6241 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     6301 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     6361 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6421 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6481 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     6541 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     6601 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     6661 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     6721 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     6781 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     6841 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     6901 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     6961 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     7021 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     7081 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     7141 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     7201 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     7261 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     7321 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     7381 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     7441 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     7501 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     7561 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     7621 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     7681 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     7741 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     7801 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
     7861 atacgactca ctatagg
//
