LOCUS       sequence-329438-        6428 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 6428)
  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 6428)
  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
FEATURES             Location/Qualifiers
     source          1..6428
                     /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"
     terminator      1254..1301
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1476..1481,1482..1498,1499..1504))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1531..1549)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1617..1721
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1722..1790,1791..2582)
                     /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(1940..1959)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2753..3341
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3242..3261
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3495..3512
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3527..3666
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3592..3611)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3752..3774
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3768..3959)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5271..6353)
                     /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     6315..6334
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(6354..6428,1..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 ctgatgtacc ggcagcggta agggaattct
      961 cccgtacact gtttaacatg cgtccagaca taactttgtt cgttagtagg acagtattca
     1021 acagcgatat gagaggtgtt ttgggcttag tgaaggtccc ttgtcatatc tttcagacag
     1081 ctagagatca ttctgtgcct gcgagtgtag ccacttatct aaagaaccac ctaggcggta
     1141 aaaatacagt gcactggctt aacatagaag gtcaccttcc acatctaagt gcgcccactc
     1201 ttttagcaca agagcttagg cgtgcactgt ctcatcgtta agctgagcaa taactagcat
     1261 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1321 ccggatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc
     1381 cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg
     1441 actgcgttag caatttaact gtgataaact accgcattaa agcttatcga tgataagctg
     1501 tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
     1561 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     1621 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1681 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1741 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1801 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1861 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1921 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     1981 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2041 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2101 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2161 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2221 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     2281 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2341 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2401 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2461 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2521 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2581 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2641 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2701 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2761 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2821 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2881 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2941 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3001 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3061 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3121 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3181 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3241 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3301 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3361 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3421 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3481 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3541 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     3601 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3661 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3721 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3781 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3841 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     3901 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3961 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     4021 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     4081 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     4141 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     4201 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     4261 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4321 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4381 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg
     4441 ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc
     4501 caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga
     4561 gtggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc
     4621 atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg
     4681 ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa
     4741 tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt
     4801 catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga
     4861 gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc
     4921 ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     4981 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     5041 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     5101 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     5161 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     5221 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     5281 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5341 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     5401 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     5461 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     5521 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     5581 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     5641 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     5701 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     5761 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     5821 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     5881 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     5941 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     6001 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     6061 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     6121 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     6181 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     6241 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     6301 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     6361 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     6421 tcgatggt
//
