LOCUS       sequence-369190-        6000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the fusion protein of miRFP2-Actin-C in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6000)
  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 6000)
  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: pmiRFP2-Actin-C-18
FEATURES             Location/Qualifiers
     source          1..6000
                     /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"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2860..2981
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2897..2916)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2951..2970
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2988..3443)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3125..3146)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3337..3356
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3470..3574
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3599..3619)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3643..3853
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        3665..3861
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3712..3847
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3896..4690
                     /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             3896..4690
                     /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(3950..3969)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4560..4579
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4878..4897)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4922..4969
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5298..5886
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5787..5806
                     /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 ccggtcgcca ccatggtcgc aggacacgca agtggaagcc ccgactttgg aaccgccagt
      661 ccgagcgact gtgagcacga agaaatccat ctggccggat ccatccagcc acacggaaca
      721 ctgctggtgg tctccgagcc tgaccataga ataattcagg catctgccaa cgccgctgaa
      781 ttcctgaatc tgggaagtgt gctgggaatc cccctggctg agatcgacgg ggatctactg
      841 atcaagattc tgccccacct ggaccttacc gcagaaggaa tgccagtggc cgtcaggtgc
      901 cgcattggaa acccctccac agagtacgac ggactgatgc atcgaccacc tgaaggagga
      961 ctgatcattg agctggaacg ggctgggcca tccattgatc tgagcggaac actggctccc
     1021 gcactggagc gaattcgaac tgcaggatcc ctgagggctc tgtgcgacga tactgtactg
     1081 ctgttccagc agtgtaccgg ctacgaccga gtgatggtct atcggttcga tgaacggggg
     1141 cacggagagg tgtattcaga aattcatgtg actggactgg agagctactt tggcaacagg
     1201 tatcctagct ccctcgtgcc acagatggcc cggcggctgt acgtgagaca gagggtgcgc
     1261 gtcctggtag atgtcaccta tcagcctgtg ccactggagc caagactgag tcccctgact
     1321 ggcagggacc tggatatgtc agggtgtttt ctgcgctcta tgagtcctac tcacctgcag
     1381 ttcctgaaga acatgggcgt gcgggcaaca ctggtggtca gcctggtggt cggaggcaaa
     1441 ctgtgggggc tggtgatctg ccaccattat ctgccacgct tcatccattt tgagctgcga
     1501 gcaatttgtg tgctgctggc cgaagctatc gcaacccgca ttacagccct gtccggactc
     1561 agatctggca gcggtggagg cagcgcatcc ggcggaagcg gaagcgatga tgatatcgcc
     1621 gcgctcgtcg tcgacaacgg ctccggcatg tgcaaggccg gcttcgcggg cgacgatgcc
     1681 ccccgggccg tcttcccctc catcgtgggg cgccccaggc accagggcgt gatggtgggc
     1741 atgggtcaga aggattccta tgtgggcgac gaggcccaga gcaagagagg catcctcacc
     1801 ctgaagtacc ccatcgagca cggcatcgtc accaactggg acgacatgga gaaaatctgg
     1861 caccacacct tctacaatga gctgcgtgtg gctcccgagg agcaccccgt gctgctgacc
     1921 gaggcccccc tgaaccccaa ggccaaccgc gagaagatga cccagatcat gtttgagacc
     1981 ttcaacaccc cagccatgta cgttgctatc caggctgtgc tatccctgta cgcctctggc
     2041 cgtaccactg gcatcgtgat ggactccggt gacggggtca cccacactgt gcccatctac
     2101 gaggggtatg ccctccccca tgccatcctg cgtctggacc tggctggccg ggacctgact
     2161 gactacctca tgaagatcct caccgagcgc ggctacagct tcaccaccac ggccgagcgg
     2221 gaaatcgtgc gtgacattaa ggagaagctg tgctacgtcg ccctggactt cgagcaagag
     2281 atggccacgg ctgcttccag ctcctccctg gagaagagct acgagctgcc tgacggccag
     2341 gtcatcacca ttggcaatga gcggttccgc tgccctgagg cactcttcca gccttccttc
     2401 ctgggcatgg agtcctgtgg catccacgaa actaccttca actccatcat gaagtgtgac
     2461 gtggacatcc gcaaagacct gtacgccaac acagtgctgt ctggcggcac caccatgtac
     2521 cctggcattg ccgacaggat gcagaaggag atcactgccc tggcacccag cacaatgaag
     2581 atcaagatca ttgctcctcc tgagcgcaag tactccgtgt ggatcggcgg ctccatcctg
     2641 gcctcgctgt ccaccttcca gcagatgtgg atcagcaagc aggagtatga cgagtccggc
     2701 ccctccatcg tccaccgcaa atgcttctag ggatccaccg gatctagata actgatcata
     2761 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     2821 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     2881 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     2941 cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt
     3001 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     3061 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     3121 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     3181 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     3241 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     3301 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     3361 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     3421 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     3481 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     3541 ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg
     3601 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3661 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3721 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3781 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3841 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
     3901 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3961 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4021 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     4081 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4141 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4201 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4261 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4321 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4381 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     4441 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4501 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4561 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4621 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4681 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4741 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4801 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4861 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     4921 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
     4981 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     5041 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     5101 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
     5161 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5221 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5281 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5341 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5401 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     5461 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5521 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5581 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5641 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5701 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5761 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5821 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5881 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5941 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
