LOCUS       sequence-371214-        6180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the fusion protein of Keratin-emiRFP2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6180)
  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 6180)
  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: pKeratin-emiRFP2-N1
FEATURES             Location/Qualifiers
     source          1..6180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     polyA_signal    2908..3029
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2945..2964)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2999..3018
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3036..3491)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3173..3194)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3385..3404
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3518..3622
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3624..3981
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3624..3815
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3647..3667)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3832..3967
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4016..4810
                     /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             4016..4810
                     /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(4070..4089)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4680..4699
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4998..5017)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5042..5089
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5418..6006
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5907..5926
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggacttttca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcacca
      541 tgagcttcac cactcgctcc accttctcca ccaactaccg gtccctgggc tctgtccagg
      601 cgcccagcta cggcgcccgg ccggtcagca gcgcggccag cgtctatgca ggcgctgggg
      661 gctctggttc ccggatctcc gtgtcccgct ccaccagctt caggggcggc atggggtccg
      721 ggggcctggc caccgggata gccgggggtc tggcaggaat gggaggcatc cagaacgaga
      781 aggagaccat gcaaagcctg aacgaccgcc tggcctctta cctggacaga gtgaggagcc
      841 tggagaccga gaaccggagg ctggagagca aaatccggga gcacttggag aagaagggac
      901 cccaggtcag agactggagc cattacttca agatcatcga ggacctgagg gctcagatct
      961 tcgcaaatac tgtggacaat gcccgcatcg ttctgcagat tgacaatgcc cgtcttgctg
     1021 ctgatgactt tagagtcaag tatgagacag agctggccat gcgccagtct gtggagaacg
     1081 acatccatgg gctccgcaag gtcattgatg acaccaatat cacacgactg cagctggaga
     1141 cagagatcga ggctctcaag gaggagctgc tcttcatgaa gaagaaccac gaagaggaag
     1201 taaaaggcct acaagcccag attgccagct ctgggttgac cgtggaggta gatgccccca
     1261 aatctcagga cctcgccaag atcatggcag acatccgggc ccaatatgac gagctggctc
     1321 ggaagaaccg agaggagcta gacaagtact ggtctcagca gattgaggag agcaccacag
     1381 tgctcaccac acagtctgct gaggttggag ctgctgagac gacgctcaca gagctgagac
     1441 gtacagtcca gtccttggag atcgacctgg actccatgag aaatctgaag gccagcttgg
     1501 agaacagcct gagggaggtg gaggcccgct acgccctaca gatggagcag ctcaacggga
     1561 tcctgctgca ccttgagtca gagctggcac agacccgggc agagggacag cgccaggccc
     1621 aggagtatga ggccctgctg aacatcaagg tcaagctgga ggctgagatc gccacctacc
     1681 gccgcctgct ggaagatggc gaggacttta atcttggtga tgccttggac agcagcaact
     1741 ccatgcaaac catccaaaag accaccaccc gccggatagt ggatggcaaa gtggtgtctg
     1801 agaccaatga caccaaagtt ctgaggcatc gaattctgca gtcgacggta ccaccggtca
     1861 tggccgaggg cagcgtggcc agacagcccg acctgctgac ctgtgagcac gaagaaatcc
     1921 atctggccgg atccatccag ccacacggaa cactgctggt ggtctccgag cctgaccata
     1981 gaataattca ggcatctgcc aacgccgctg aattcctgaa tctgggaagt gtgctgggaa
     2041 tccccctggc tgagatcgac ggggatctac tgatcaagat tctgccccac ctggacctta
     2101 ccgcagaagg aatgccagtg gccgtcaggt gccgcattgg aaacccctcc acagagtacg
     2161 acggactgat gcatcgacca cctgaaggag gactgatcat tgagctggaa cgggctgggc
     2221 catccattga tctgagcgga acactggctc ccgcactgga gcgaattcga actgcaggat
     2281 ccctgagggc tctgtgcgac gatactgtac tgctgttcca gcagtgtacc ggctacgacc
     2341 gagtgatggt ctatcggttc gatgaacggg ggcacggaga ggtgtattca gaaattcatg
     2401 tgactggact ggagagctac tttggcaaca ggtatcctag ctccctcgtg ccacagatgg
     2461 cccggcggct gtacgtgaga cagagggtgc gcgtcctggt agatgtcacc tatcagcctg
     2521 tgccactgga gccaagactg agtcccctga ctggcaggga cctggatatg tcagggtgtt
     2581 ttctgcgctc tatgagtcct actcacctgc agttcctgaa gaacatgggc gtgcgggcaa
     2641 cactggtggt cagcctggtg gtcggaggca aactgtgggg gctggtgatc tgccaccatt
     2701 atctgccacg cttcatccat tttgagctgc gagcaatttg tgtgctgctg gccgaagcta
     2761 tcgcaacccg cattacagcc ctgtaggcgg ccgcgactct agatcataat cagccatacc
     2821 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     2881 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     2941 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     3001 ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt
     3061 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     3121 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     3181 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     3241 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     3301 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     3361 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3421 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3481 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3541 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3601 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     3661 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3721 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3781 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3841 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3901 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3961 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
     4021 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4081 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4141 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     4201 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4261 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4321 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4381 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4441 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4501 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     4561 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4621 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4681 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4741 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4801 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4861 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4921 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4981 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     5041 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
     5101 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     5161 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     5221 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
     5281 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5341 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5401 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5461 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5521 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     5581 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5641 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5701 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5761 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5821 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5881 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5941 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6001 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6061 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
     6121 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
//
