LOCUS       sequence-368808-        4714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TagRFP658 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4714)
  AUTHORS   Babakhanova S, Jung EE, Namikawa K, Zhang H, Wang Y, Subach OM, 
            Korzhenevskiy DA, Rakitina TV, Xiao X, Wang W, Shi J, Drobizhev M, 
            Park D, Eisenhard L, Tang H, Koster RW, Subach FV, Boyden ES, 
            Piatkevich KD
  TITLE     Rapid directed molecular evolution of fluorescent proteins in 
            mammalian cells.
  JOURNAL   Protein Sci. 2022 Mar;31(3):728-751. doi: 10.1002/pro.4261. Epub 
            2021 Dec 30.
  PUBMED    34913537
REFERENCE   2  (bases 1 to 4714)
  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: "10.1002/pro.4261"; 
            journalName: "Protein Sci"; date: "2022-03"; volume: "31"; issue: 
            "3"; pages: "728-751"
COMMENT     Sequence Label: pTagRFP658-N1
FEATURES             Location/Qualifiers
     source          1..4714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    591..671
                     /label=MCS
                     /note="multiple cloning site"
     CDS             679..1380
                     /codon_start=1
                     /product="far-red monomeric fluorescent protein with an 
                     emission peak at 657 nm (Morozova et al., 2010)"
                     /label=TagRFP657
                     /note="derived from mKate"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTQRIKV
                     VEGGPLPFAFDILATSFMYGSHTFIDHTQGIPDFWKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLHDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEAHTEMLYPTDGGLEGRTALALK
                     LVGGGHLICNFKTIYRSKKPAKNLKMPGVYYVDYRLERIEEADNETYVEQHEVAVARYC
                     DLPTKLGHKLN"
     polyA_signal    1502..1623
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1539..1558)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1593..1612
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1630..2085)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1767..1788)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1979..1998
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2112..2216
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2218..2575
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2218..2409
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2241..2261)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2426..2561
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2610..3404
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2610..3404
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2664..2683)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3274..3293
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3592..3611)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3636..3683
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4012..4600
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4501..4520
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccaccgg tcgccaccat gagcgagctg attaaggaga acatgcacat gaagctgtac
      721 atggagggca ccgtgaacaa tcaccacttc aagtgcacat cagagggcga aggcaagccc
      781 tacgagggca cccagaccca gagaatcaag gtggtcgagg gcggccctct ccccttcgcc
      841 ttcgacatcc tggctaccag cttcatgtac ggcagtcaca ccttcatcga ccacacccag
      901 ggcatccccg acttctggaa gcagtccttc cctgagggct tcacgtggga gagagtcacc
      961 acatacgaag acgggggcgt cctgaccgct acccaggaca ccagcctcca tgacggctgc
     1021 ctcatctaca acgtcaagat cagaggggtg aacttcccat ccaacggccc tgtgatgcag
     1081 aagaaaacac tcggctggga ggcccacacc gagatgttgt accccactga cggcggtctg
     1141 gaaggcagaa ccgcgctggc cctgaagctc gtgggcgggg gccacttaat ctgcaacttc
     1201 aagactatat acagatccaa gaaacccgct aagaacctca agatgcccgg cgtctactat
     1261 gtggactaca gactggaaag aatcgaggag gccgacaatg agacctacgt cgagcagcac
     1321 gaggtggctg tggccagata ctgcgacctc cctaccaaac tggggcacaa gcttaattaa
     1381 gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     1441 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     1501 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     1561 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     1621 ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     1681 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     1741 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     1801 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     1861 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     1921 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     1981 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     2041 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     2101 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2161 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     2221 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     2281 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     2341 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2401 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     2461 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     2521 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     2581 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     2641 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     2701 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     2761 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     2821 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     2881 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     2941 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3001 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3061 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3121 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3181 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3241 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3301 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3361 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     3421 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     3481 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     3541 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     3601 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     3661 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     3721 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     3781 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     3841 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     3901 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     3961 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4021 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4081 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4141 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4201 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4261 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4321 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4381 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4441 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4501 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4561 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4621 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4681 tgattctgtg gataaccgta ttaccgccat gcat
//
