LOCUS       sequence-101267-        5374 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Duet vector for co-expression of GFP and mCherry from PBAD and 
            PLtetO promoters, respectively; parent vector for split mCherry 
            assembly vectors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5374)
  AUTHORS   Sawyer N, Gassaway BM, Haimovich AD, Isaacs FJ, Rinehart J, Regan L
  TITLE     Designed Phosphoprotein Recognition in Escherichia coli.
  JOURNAL   ACS Chem Biol. 2014 Nov 21;9(11):2502-7. doi: 10.1021/cb500658w. 
            Epub 2014 Oct 6.
  PUBMED    25272187
REFERENCE   2  (bases 1 to 5374)
  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/cb500658w";
            journalName: "ACS Chem Biol"; date: "2014-11-21- 21"; volume: "9"; 
            issue: "11"; pages: "2502-7"
COMMENT     Sequence Label: pNAS1B
FEATURES             Location/Qualifiers
     source          1..5374
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(144..161)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(278..823)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(358..377)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(971..1849)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1876..2160
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     2083..2102
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             2195..2908
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2225..2253)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2848..2868
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2952..2969)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3122..3208
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3300..3327
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3357..3420
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3421..3489,3490..4281)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3639..3658)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        join(4427..4445,4446..4451,4452..4468,4469..4474,
                     4475..4500)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    4427..4445
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4452..4470
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             4510..4521
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4537..5244
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(4679..4697)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4947..4966
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      5278..5364
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cctaggatca gcgctagcgg agtgtatact ggcttactat gttggcactg atgagggtgt
       61 cagtgaagtg cttcatgtgg caggagaaaa aaggctgcac cggtgcgtca gcagaatatg
      121 tgatacagga tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg
      181 cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata
      241 cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg
      301 acaagcatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa
      361 gataccaggc gtttccccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt
      421 ttaccggtgt cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt
      481 tccgggtagg cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac
      541 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca
      601 ccactggcag cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg
      661 ttaaggctaa actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg
      721 gttcaaagag ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc
      781 gttttcagag caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa
      841 ctgcagagat caattcgcgc gcgaaggcga agcggcatgc tgcataatgt gcctgtcaaa
      901 tggacgaagc agggattctg caaaccctat gctactccgt caagccgtca attgtctgat
      961 tcgttaccaa ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac
     1021 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat
     1081 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca
     1141 gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct
     1201 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga
     1261 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat
     1321 tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct
     1381 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga
     1441 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg
     1501 tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt
     1561 aaacccactg gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc
     1621 ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca
     1681 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt
     1741 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg
     1801 cattaaacga gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac
     1861 tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg
     1921 tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt
     1981 aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca
     2041 gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat
     2101 ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat
     2161 acccgttttt tgggctaaca ggaggaatga cgtcatgagt aaaggagaag aacttttcac
     2221 tggagttgtc ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt
     2281 cagtggagag ggtgaaggtg atgcaacata cggaaaactt acccttaaat ttatttgcac
     2341 tactggaaaa ctacctgttc catggccaac acttgtcact actttcggtt atggtgttca
     2401 atgctttgcg agatacccag atcatatgaa acagcatgac tttttcaaga gtgccatgcc
     2461 cgaaggttat gtacaggaaa gaactatatt tttcaaagat gacgggaact acaagacacg
     2521 tgctgaagtc aagtttgaag gtgataccct tgttaataga atcgagttaa aaggtattga
     2581 ttttaaagaa gatggaaaca ttcttggaca caaattggaa tacaactata actcacacaa
     2641 tgtatacatc atggcagaca aacaaaagaa tggaatcaaa gttaacttca aaattagaca
     2701 caacattgaa gatggaagcg ttcaactagc agaccattat caacaaaata ctccaattgg
     2761 cgatggccct gtccttttac cagacaacca ttacctgtcc acacaatctg ccctttcgaa
     2821 agatcccaac gaaaagagag accacatggt ccttcttgag tttgtaacag ctgctgggat
     2881 tacacatggc atggatgaac tctacaaata agagctcttg gctgttttgg cggatgagag
     2941 aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat
     3001 ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa
     3061 cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca
     3121 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     3181 ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca
     3241 acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca
     3301 gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcctcgag ttcaaatatg
     3361 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3421 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3481 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3541 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3601 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3661 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3721 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3781 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3841 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3901 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3961 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     4021 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     4081 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4141 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4201 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4261 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4321 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4381 accaaaatcc cttaacgtga gttttcgttc cactgagcga agatcttccc tatcagtgat
     4441 agagattgac atccctatca gtgatagaga tactgagcac atcagcagga cgcactgacc
     4501 gaattcatta aagaggagaa aggtaccatg gcatccgtga gcaagggcga ggaggataac
     4561 atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc
     4621 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     4681 aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag
     4741 ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag
     4801 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg
     4861 gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg
     4921 cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag
     4981 gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg
     5041 ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag
     5101 aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac
     5161 aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc
     5221 ggcatggacg agctgtacaa gtgataaaag cttcatggta cgcgtgctgc caggcatcaa
     5281 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     5341 aacgctctcc tgagtaggac aaatccgccg ccct
//
