LOCUS       sequence-343313-        6212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Rationally enhanced red fluorescent protein derived from TagRFP-T. 
            For mammalian expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6212)
  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 6212)
  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-StagRFP
FEATURES             Location/Qualifiers
     source          1..6212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        38..417
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        418..621
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     572..592
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     666..685
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        666..684
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      704..713
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             725..742
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             746..778
                     /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(782..790,791..805)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             812..1507
                     /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(1568..1586)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1569..1586)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1606..1623)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1612..1836
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1882..2310
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1969..1988)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2179..2200
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2319..2339)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2324..2653
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2504..2639
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2566..2585
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2720..3514
                     /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(2774..2793)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3384..3403
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3688..3809
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3725..3744)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3779..3798
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3858..3874)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3858..3874)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3871..3893)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3882..3898
                     /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(3906..3912,3913..3930,3931..3936))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3951..3972
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4089..4106)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4260..4848)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4340..4359)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5019..5810,5811..5879))
                     /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     5642..5661
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5880..5984)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6059..6078)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat
       61 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      121 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      181 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      241 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      301 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      361 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      421 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      481 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      541 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
      601 gaggtctata taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc
      661 gaaattaata cgactcacta tagggagacc caagcttgcg gccgccacca tggtgcgggg
      721 ttctcatcat catcatcatc atggtatggc tagcatgact ggtggacagc aaatgggtcg
      781 ggatctgtac gacgatgacg ataaggatcc gatgagcgag ctgattaagg agaacatgca
      841 catgaagctg tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg
      901 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc
      961 tctccccttc gccttcgaca tcctggctac cagcttcatg tacggcagca gaaccttcat
     1021 caaccacacc cagggcatcc ccgatttctt taagcagtcc ttccctgagg gcttcacatg
     1081 ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct
     1141 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg
     1201 ccctgtgatg cagaagaaaa cactcggctg ggaggccaac accgagatgc tgtaccccgc
     1261 tgacggcggc ctggaaggca gaaccgtcat ggccctgaag ctcgtgggcg ggggccacct
     1321 gatctgcaac ttcaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc
     1381 cggcgtctac tatgtggacc acagactgga aagaatcaag gaggccgaca aagagaccta
     1441 cgtcgagcag cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca
     1501 caagtaagaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg catctagagg
     1561 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt
     1621 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     1681 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     1741 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     1801 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     1861 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1921 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1981 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2041 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2101 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2161 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2221 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2281 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     2341 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2401 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2461 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     2521 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     2581 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     2641 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     2701 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     2761 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     2821 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     2881 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     2941 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     3001 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     3061 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     3121 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     3181 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     3241 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     3301 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     3361 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     3421 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     3481 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     3541 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     3601 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     3661 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     3721 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     3781 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     3841 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3901 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3961 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4021 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4081 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4141 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4201 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4261 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4321 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4381 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4441 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4501 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4561 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4621 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4681 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4741 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4801 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4861 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4921 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4981 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5041 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5101 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5161 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5221 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5281 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5341 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5401 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5461 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5521 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5581 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5641 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5701 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5761 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5821 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5881 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5941 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6001 gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta
     6061 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     6121 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     6181 gcatgaagaa tctgcttagg gttaggcgtt tt
//
