LOCUS       sequence-111219-        6139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRET biosensor Destination vector for E. coli expression. Encodes a 
            FRET pair for fusion with ligand binding domain of interest..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6139)
  AUTHORS   Jones AM, Danielson JA, Manojkumar SN, Lanquar V, Grossmann G, 
            Frommer WB
  TITLE     Abscisic acid dynamics in roots detected with genetically encoded 
            FRET sensors.
  JOURNAL   eLife. 2014 Apr 15;3:e01741. doi: 10.7554/eLife.01741.
  PUBMED    24737862
REFERENCE   2  (bases 1 to 6139)
  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"; journalName: "eLife."; 
            date: "2014-04-15"; volume: "3:e01741. doi"; pages: " 
            10.7554/eLife.01741"
COMMENT     Sequence Label: pFLIP30
FEATURES             Location/Qualifiers
     source          1..6139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..38
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        19..37
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             69..91
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             111..128
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             132..164
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(168..176,177..191)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     protein_bind    906..1030
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1055..1060,1061..1078,1079..1085)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(1205..1224)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2198..2503
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2382..2401
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2544..2668)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(2682..2684,2685..3395)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2724..2745)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2985..3004)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3332..3353
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3396..3425
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3484..3502)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3498..3545
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3642..4097
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3729..3748)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3939..3960
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4178..4282
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4283..4351,4352..5143)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCDTLLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDESDTTMPVAMPTTLRKLLTGELLTLASRQQLID*MEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKRSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5314..5902
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5803..5822
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6056..6073
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta
       61 gaaataattt tgtttaactt taagaaggag atatacatat gcggggttct catcatcatc
      121 atcatcatgg tatggctagc atgactggtg gacagcaaat gggtcgggat ctgtacgacg
      181 atgacgataa ggatccgggc ctcgaggttt ctaaaggtga agaattgttt acgggcgtcg
      241 tcccgatcct cgtggaactc gacggggatg ttaacgggca taagttttcg gtcagcgggg
      301 aaggggaggg ggacgcgacg tatgggaagc tcactctcaa gctgatctgt acgacgggga
      361 aactcccggt cccgtggccg acgctggtca cgacgctggg atacgggctc caatgctttg
      421 cgaggtatcc ggaccacatg aaacagcatg actttttcaa atcggcgatg ccggagggat
      481 acgtgcagga acggacgatc tttttcaaag acgatgggaa ctataagacg cgggcggaag
      541 tcaagtttga aggggacacg ctcgtcaacc ggatcgaact caaggggatt gacttcaaag
      601 aggatgggaa catactcggc cataagctcg aatacaatta caactcgcat aacgtataca
      661 tcaccgcgga taagcaaaag aacgggatca aagccaattt caaaatccgg cataacatag
      721 aggatggggg ggtccaactg gcggatcact atcagcaaaa cacgccgata ggggatgggc
      781 cggtcctcct cccggataac cattacctct cgtaccaaag cgcgctctcg aaggacccga
      841 atgagaaacg ggaccacatg gttctcctgg agttcgtcac ggcggcgggc ataggtaccg
      901 atatcacaag tttgtacaaa aaagctgaac gagaaacgta aaatgatata aatatcaata
      961 tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca caacatatcc
     1021 agtcactatg gcggccgcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     1081 taatgtgtgg attttgagtt aggatccgtc gagattttca ggagctaagg aagctaaaat
     1141 ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca
     1201 ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat
     1261 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
     1321 cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga aagacggtga
     1381 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac
     1441 gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc
     1501 gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa
     1561 tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc
     1621 caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga
     1681 caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg ggcttccatg
     1741 tcggcagaat gcttaatgaa ttacacagta ctgcgatgag tggcagggcg gggcgtaaac
     1801 gcgtggatcc ggcttactaa aagccagata acagtatgcg tatttgcgcg ctgatttttg
     1861 cggtataaga atatatactg atatgtatac ccgaagtatg tcaaaaagag gtatgctatg
     1921 aagcagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa ggcatatatg
     1981 atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt cgtctgcgtg
     2041 ccgaacgctg gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg tttattgaaa
     2101 tgaacggctc ttttgctgac gagaacaggg gctggtgaaa tgcagtttaa ggtttacacc
     2161 tataaaactt ttgctgacga gaacaggggc tggtgaaatg cagtttaagg tttacaccta
     2221 taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc
     2281 cgggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg
     2341 tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat
     2401 ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa
     2461 tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag gctcccttat
     2521 acacagccag tctgcaggtc gaccatagtg actggatatg ttgtgtttta cagtattatg
     2581 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
     2641 ctcgttcagc tttcttgtac aaagtggtga tatcgactag tgtgagcaag ggcgaggagc
     2701 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     2761 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     2821 tctgcaccac cggtaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctggg
     2881 gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2941 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     3001 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     3061 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aacgccatca
     3121 gcgacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     3181 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     3241 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccaagc
     3301 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     3361 ccgggatcac tctcggcatg gacgagctgt acaaggaaca aaaattgata agtgaggaag
     3421 atttataagg gcccgatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     3481 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     3541 ttttgctgaa aggaggaact atatccggat ctggcgtaat agcgaagagg cccgcaccga
     3601 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     3661 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3721 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3781 tcaagctcta aatcgggggc tccctttagg gttccgattt agagctttac ggcacctcga
     3841 ccgcaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3901 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3961 aacaacactc aaccctatcg cggtctattc ttttgattta taagggattt tgccgatttc
     4021 ggcctattgg ttaaaaaatg agctgattta acaaatattt aacgcgaatt ttaacaaaat
     4081 attaacgttt acaatttcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4141 cacaccgcat acaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     4201 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4261 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     4321 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     4381 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     4441 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     4501 gctatgtgat acactattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     4561 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     4621 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     4681 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     4741 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     4801 cgacgagagt gacaccacga tgcctgtagc aatgccaaca acgttgcgca aactattaac
     4861 tggcgaacta cttactctag cttcccggca acaattaata gactgaatgg aggcggataa
     4921 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     4981 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcg
     5041 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5101 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     5161 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5221 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5281 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5341 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5401 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5461 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5521 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5581 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5641 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5701 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5761 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5821 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5881 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctgggctt
     5941 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     6001 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     6061 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     6121 gccgattcat taatgcagg
//
