LOCUS       sequence-387257-        6679 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian/zebrafish expression of zmiRFP-EF1-2xCMV-EF1-mClover3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6679)
  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 6679)
  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: pTol2zmiRFP-EF1-2xCMV-EF1-mClover3
FEATURES             Location/Qualifiers
     source          1..6679
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(30..41,42..179)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     promoter        187..205
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    323..457
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      1457..1466
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3289..3291,3292..3294,3295..4008)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(3334..3355)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3942..3963
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4039..4173
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(4276..4294)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    join(4301..4488,4489..4500)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4530..4546)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4530..4546)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4543..4565)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4554..4570
                     /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(4578..4584,4585..4602,4603..4608))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4623..4644
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4761..4778)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4932..5520)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5012..5031)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5691..6482,6483..6551))
                     /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     6314..6333
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6552..6656)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaatc ccatatgggc agaggtgtaa aaagtactca aaaattttac
       61 tcaagtgaaa gtacaagtac ttagggaaaa ttttactcaa ttaaaagtaa aagtatctgg
      121 ctagaatctt acttgagtaa aagtaaaaaa gtactccatt aaaattgtac ttgagtatta
      181 gatctgtaat acgactcact atagggacat agaatgcagg taccgggccc aatgcattgg
      241 cgccgcggcc ggccgcgaat taaaaaacct cccacacctc cccctgaacc tgaaacataa
      301 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
      361 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
      421 gtccaaactc atcaatgtat cttatcatgt ctggatctac gtaatacgac tcactatagt
      481 tctagcctcg agaaggccgg ccataagatc taatggcgcg cctcacagag ctgtgattct
      541 tgtagcgata gcctcagcca gcagctcaca gatagctctc agctcgaagt ggatgaatct
      601 aggcaggtag tggtgacaga tcaccagtcc ccacagcttt cctcccacca ccaggctcac
      661 caccagtgta gctctcactc ccatgttctt caggaactgc aggtgtgtag ggctcatgct
      721 tctcaggaaa catccgctca tgtccaggtc tcttcctgtc agagggctca gtctaggctc
      781 cagaggcaca ggctggtagc tcacgtccac cagcactctc actctctgtc tctcgtacag
      841 tcttctagcc atctgaggca ccaggctgct agggtatctg tttccgaagt agctctccag
      901 tcctgtcacg tggatctcgc tgtacacctc tccgtgtccc tgctcgtcga atctgtacac
      961 catcactctg tcgtatcctg tacactgctg gaacagcaga gctgtgtcgt cacacagagc
     1021 tctcaggctt ccagctgttc tgattctctc cagagcagga gccagtgttc cgctcaggtc
     1081 gataggaggt ccagctctct ccagctcgat gatcagtcct ccctcaggag gtctgtgcat
     1141 cagtccgtcg tactctgtgc tagggtttcc gattctacat ctcacagcca caggcattcc
     1201 ctcagctgta gggtccaggt gaggcaggat cttgatcagc aggtctccgt cgatctcagc
     1261 cagaggcact cccagcacgc ttcccaggtt caggaactca gcagcgttag cgctagcctg
     1321 gatgattctg tggtcaggct cgctcaccac cagcagtgtt ccgtgaggct ggatgcttcc
     1381 agccaggtgg atctcctctc tctcacagtc gctagggtca gctgttccga agtcagggct
     1441 tccgctagcg tgtccagcca ccatggtgga tatcgtttaa acaattctgg aagctactgt
     1501 acaccaacct gtcaggagag gaaagagaag aaggttagta caattgtcta gggctgcagg
     1561 gttcacagtg ccacttttcc tgcactgccc cacctcctgc ccaccctttc ccaggcatag
     1621 acagtcagtg acttaccaaa ctcacaggag ggagaaggca gaagcttgaa tgttcacaga
     1681 gactactgca cttatatatg gttctccccc accctgggga aaaaggtgga gccagtacac
     1741 cacatcactt tcccagttta cccaagcccc accttctcta ggcaccagtt caattgccca
     1801 cccctccccc caacttctca gggactgtgg gccatgtgct ctctgcccac tgaggggcac
     1861 tcagccctca agcatgctct tctccactag tcacccctat tgaccttatg tatgtgccaa
     1921 taatgggaaa aacccattga ctcaccccct attgaccttt tgtactgggc aaaacccaat
     1981 ggaaagtccc tattgactca gtgtacttgg ctccaatggg actttcctgt tgattcaccc
     2041 ctattgacct tatgtactgg gcaaaaccca ttggaaagtc cctaatgact cagtatacat
     2101 gtcagtaatg ggaaaaaccc attggcttac ctcccattga ccttatgtac tgggcaaaac
     2161 ccattggaaa gtccctattg actcaatgta cttggctcca atgggacttt cctgttgact
     2221 caccccctat tgaccttatg tactgggcaa aacccaatgg aaagtcccta ttgagtcagt
     2281 gtacttggct ccaatgggtt tttcccattg actcctgcat taatcgattt aaatgctagg
     2341 gctgctctcc aatctctgtc atggcagggc cactggcatc cctgaagttg gtggagacca
     2401 cctcactcca ctcagcatac agctcatcca gtcctcttaa ttaatgcagg agtcaatggg
     2461 aaaaacccat tggagccaag tacactgact caatagggac tttccattgg gttttgccca
     2521 gtacataagg tcaatagggg gtgagtcaac aggaaagtcc cattggagcc aagtacattg
     2581 agtcaatagg gactttccaa tgggttttgc ccagtacata aggtcaatgg gaggtaagcc
     2641 aatgggtttt tcccattact gacatgtata ctgagtcatt agggactttc caatgggttt
     2701 tgcccagtac ataaggtcaa taggggtgaa tcaacaggaa agtcccattg gagccaagta
     2761 cactgagtca atagggactt tccattgggt tttgcccagt acaaaaggtc aatagggggt
     2821 gagtcaatgg gtttttccca ttattggcac atacataagg tcaatagggg tgactagtgg
     2881 agaagagcat gcttgagggc tgagtgcccc tcagtgggca gagagcacat ggcccacagt
     2941 ccctgagaag ttggggggag gggtgggcaa ttgaactggt gcctagagaa ggtggggctt
     3001 gggtaaactg ggaaagtgat gtggtgtact ggctccacct ttttccccag ggtgggggag
     3061 aaccatatat aagtgcagta gtctctgtga acattcaagc ttctgccttc tccctcctgt
     3121 gagtttggta agtcactgac tgtctatgcc tgggaaaggg tgggcaggag gtggggcagt
     3181 gcaggaaaag tggcactgtg aaccctgcag ccctagacaa ttgtactaac cttcttctct
     3241 ttcctctcct gacaggttgg tgtacagtag cttccagaat tcgcaaacat ggtgagcaag
     3301 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     3361 ggccacaagt tcagcgtccg cggcgagggc gagggcgatg ccaccaacgg caagctgacc
     3421 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     3481 ttcggctacg gcgtggcctg cttcagccgc taccccgacc acatgaagca gcacgacttc
     3541 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatctcttt caaggacgac
     3601 ggcacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     3661 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     3721 aacttcaaca gccactacgt ctatatcacg gccgacaagc agaagaactg catcaaggct
     3781 aacttcaaga tccgccacaa cgttgaggac ggcagcgtgc agctcgccga ccactaccag
     3841 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagccat
     3901 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     3961 gtgaccgccg ccgggattac acatggcatg gacgagctgt acaagtaaat ctagaactat
     4021 agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc
     4081 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4141 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4201 tttcaggttc agggggaggt gtgggaggtt ttttaattcg cggccgccac cgcggtggag
     4261 ctccagcttt tgttcccttt agtgagggtt aattgggccc aagtgatctc caaaaaataa
     4321 gtactttttg actgtaaata aaattgtaag gagtaaaaag tacttttttt tctaaaaaaa
     4381 tgtaattaag taaaagtaaa agtattgatt tttaattgta ctcaagtaaa gtaaaaatcc
     4441 ccaaaaataa tacttaagta cagtaatcaa gtaaaattac tcaagtactt tacacctctg
     4501 ggtactgttt cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4561 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4621 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4681 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4741 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4801 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4861 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4921 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4981 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5041 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5101 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5161 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5221 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5281 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5341 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5401 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5461 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5521 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5581 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5641 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5701 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5761 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5821 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5881 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     5941 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6001 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6061 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6121 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6181 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6241 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6301 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6361 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6421 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6481 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6541 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6601 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6661 tttccccgaa aagtgccac
//
