LOCUS       sequence-344975-        7319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Long-Stokes-shift green-red FRET-based Src activity reporter for 
            multiplexed imaging applications..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7319)
  AUTHORS   Mo GCH, Posner C, Rodriguez EA, Sun T, Zhang J
  TITLE     A rationally enhanced red fluorescent protein expands the utility of
            FRET biosensors.
  JOURNAL   Nat Commun. 2020 Apr 15;11(1):1848. doi: 10.1038/s41467-020-15687-x.
  PUBMED    32296061
REFERENCE   2  (bases 1 to 7319)
  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: "Nat 
            Commun."; date: "2020-04-15"; volume: "11(1):1848. doi"; pages: " 
            10.1038/s41467-020-15687-x"
COMMENT     Sequence Label: pcDNA3-Tsapphire-StagRFP-SrcAR
FEATURES             Location/Qualifiers
     source          1..7319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(75..94)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        266..645
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        646..849
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     800..820
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     894..913
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        894..912
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      932..941
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             953..970
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             974..1006
                     /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(1010..1018,1019..1033)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(1040..1042,1043..1045,1046..1756)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVMVFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSIQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1085..1106)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1346..1365)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1693..1714
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2147..2842
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTVMALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHK"
     promoter        complement(2903..2921)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2904..2921)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2941..2958)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2947..3171
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3217..3645
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3304..3323)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3514..3535
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3654..3674)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3659..3988
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3839..3974
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3901..3920
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4055..4849
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4109..4128)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4719..4738
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5023..5144
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5060..5079)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5114..5133
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5193..5209)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5193..5209)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5206..5228)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5217..5233
                     /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)."
     promoter        complement(join(5241..5247,5248..5265,5266..5271))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5286..5307
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5424..5441)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5595..6183)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5675..5694)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6354..7145,7146..7214))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6977..6996
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7215..7319)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
       61 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
      121 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
      181 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
      241 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
      301 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
      361 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      421 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      481 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
      541 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
      601 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
      661 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
      721 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      781 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      841 agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg
      901 actcactata gggagaccca agcttgcggc cgccaccatg gtgcggggtt ctcatcatca
      961 tcatcatcat ggtatggcta gcatgactgg tggacagcaa atgggtcggg atctgtacga
     1021 cgatgacgat aaggatccca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1081 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1141 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1201 gcccgtgccc tggcccaccc tcgtgaccac cttctcctac ggcgtgatgg tgttcgcccg
     1261 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1321 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1381 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1441 cggcaacatc ctggggcaca agctggagta caacttcaac agccacaacg tctatatcat
     1501 ggccgacaag cagaagaacg gcatcaaggc caacttcaag atccgccaca acatcgagga
     1561 cggcggcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1621 gctgctgccc gacaaccact acctgagcat ccagtccgcc ctgagcaaag accccaacga
     1681 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     1741 ggacgagctg tacaagcgca tgcattggta ttttgggaag atcactcgtc gggagtccga
     1801 gcggctgctg ctcaaccccg aaaacccccg gggaaccttc ttggtccggg agagcgagac
     1861 gacaaaaggt gcctattgcc tctccgtttc tgactttgac aacgccaagg ggctcaatgt
     1921 gaagcactac aagatccgca agctggacag cggcggcttc tacatcacct cacgcacaca
     1981 gttcagcagc ctgcagcagc tggtggccta ctactccaaa catgctgatg gcttgtgcca
     2041 ccgcctgact aacgtctgtg ggtctacatc tggatctggg aagccgggtt ctggtgaggg
     2101 ttcttggatg gaggactatg actacgtcca cctacagggg gagctcatga gcgagctgat
     2161 taaggagaac atgcacatga agctgtacat ggagggcacc gtgaacaacc accacttcaa
     2221 gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggt
     2281 ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctaccagct tcatgtacgg
     2341 cagcagaacc ttcatcaacc acacccaggg catccccgat ttctttaagc agtccttccc
     2401 tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac
     2461 ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa
     2521 cttcccatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg ccaacaccga
     2581 gatgctgtac cccgctgacg gcggcctgga aggcagaacc gtcatggccc tgaagctcgt
     2641 gggcgggggc cacctgatct gcaacttcaa gaccacatac agatccaaga aacccgctaa
     2701 gaacctcaag atgcccggcg tctactatgt ggaccacaga ctggaaagaa tcaaggaggc
     2761 cgacaaagag acctacgtcg agcagcacga ggtggctgtg gccagatact gcgacctccc
     2821 tagcaaactg gggcacaagt aagaattctg cagatatcca tcacactggc ggccgctcga
     2881 gcatgcatct agagggccct attctatagt gtcacctaaa tgctagagct cgctgatcag
     2941 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3001 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3061 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     3121 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     3181 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     3241 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3301 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3361 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3421 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3481 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3541 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3601 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     3661 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3721 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     3781 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     3841 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     3901 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     3961 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     4021 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     4081 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     4141 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     4201 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     4261 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     4321 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     4381 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     4441 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     4501 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     4561 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4621 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4681 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4741 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4801 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4861 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4921 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4981 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     5041 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5101 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     5161 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5221 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5281 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5341 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5401 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5461 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5521 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5581 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5641 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5701 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5761 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5821 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5881 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5941 gcagcagcca ctggtaacag gattagcaaa gcgaggtatg taggcggtgc tacagagttc
     6001 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6061 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6121 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6181 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6241 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6301 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6361 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6421 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6481 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6541 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6601 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6661 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6721 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6781 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6841 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6901 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6961 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7021 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7081 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7141 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7201 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7261 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcg
//
