LOCUS       sequence-386832-        6445 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian/zebrafish expression of zTagRFP658-EF1-2xCMV-EF1-mClover3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6445)
  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 6445)
  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: pTol2zTagRFP658-EF1-2xCMV-EF1-mClover3
FEATURES             Location/Qualifiers
     source          1..6445
                     /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"
     CDS             join(3055..3057,3058..3060,3061..3774)
                     /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(3100..3121)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3708..3729
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3805..3939
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(4042..4060)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    join(4067..4254,4255..4266)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4296..4312)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4296..4312)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4309..4331)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4320..4336
                     /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(4344..4350,4351..4368,4369..4374))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4389..4410
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4527..4544)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4698..5286)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4778..4797)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5457..6248,6249..6317))
                     /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     6080..6099
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6318..6422)
                     /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 cctcagttca gcttgtgtcc
      541 cagctttgta ggcaggtcac agtatctagc cacagccacc tcgtgctgct ccacgtatgt
      601 ctcgttgtca gcctcctcga ttctctccag tctgtagtcc acgtagtaca ctccaggcat
      661 cttcaggttc ttagcaggct tcttgcttct gtagattgtc ttgaagttac agatcaggtg
      721 tcctcctccc accagcttca gagccagagc tgttcttccc tccagtcctc cgtctgtagg
      781 gtacagcatc tctgtgtgag cctcccatcc cagtgtcttc ttctgcatca caggtccgtt
      841 gctagggaag ttcactcctc tgatcttcac gttgtagatc agacatccgt cgtgcaggct
      901 tgtgtcctgt gtagctgtca gcactcctcc gtcctcgtat gttgtcactc tctcccatgt
      961 gaatccctca gggaagctct gcttccagaa gtcagggatt ccctgtgtgt ggtcgatgaa
     1021 tgtgtggctt ccgtacatga agcttgtagc caggatgtcg aaagcgaaag gcagaggtcc
     1081 tccctccacc accttgattc tctgtgtctg tgttccctcg taaggctttc cctctccctc
     1141 gcttgtacac ttgaagtggt ggttgttcac tgttccctcc atgtacagct tcatgtgcat
     1201 gttctcctta atcagctcgc tcatacccat ggtggatatc gtttaaacaa ttctggaagc
     1261 tactgtacac caacctgtca ggagaggaaa gagaagaagg ttagtacaat tgtctagggc
     1321 tgcagggttc acagtgccac ttttcctgca ctgccccacc tcctgcccac cctttcccag
     1381 gcatagacag tcagtgactt accaaactca caggagggag aaggcagaag cttgaatgtt
     1441 cacagagact actgcactta tatatggttc tcccccaccc tggggaaaaa ggtggagcca
     1501 gtacaccaca tcactttccc agtttaccca agccccacct tctctaggca ccagttcaat
     1561 tgcccacccc tccccccaac ttctcaggga ctgtgggcca tgtgctctct gcccactgag
     1621 gggcactcag ccctcaagca tgctcttctc cactagtcac ccctattgac cttatgtatg
     1681 tgccaataat gggaaaaacc cattgactca ccccctattg accttttgta ctgggcaaaa
     1741 cccaatggaa agtccctatt gactcagtgt acttggctcc aatgggactt tcctgttgat
     1801 tcacccctat tgaccttatg tactgggcaa aacccattgg aaagtcccta atgactcagt
     1861 atacatgtca gtaatgggaa aaacccattg gcttacctcc cattgacctt atgtactggg
     1921 caaaacccat tggaaagtcc ctattgactc aatgtacttg gctccaatgg gactttcctg
     1981 ttgactcacc ccctattgac cttatgtact gggcaaaacc caatggaaag tccctattga
     2041 gtcagtgtac ttggctccaa tgggtttttc ccattgactc ctgcattaat cgatttaaat
     2101 gctagggctg ctctccaatc tctgtcatgg cagggccact ggcatccctg aagttggtgg
     2161 agaccacctc actccactca gcatacagct catccagtcc tcttaattaa tgcaggagtc
     2221 aatgggaaaa acccattgga gccaagtaca ctgactcaat agggactttc cattgggttt
     2281 tgcccagtac ataaggtcaa tagggggtga gtcaacagga aagtcccatt ggagccaagt
     2341 acattgagtc aatagggact ttccaatggg ttttgcccag tacataaggt caatgggagg
     2401 taagccaatg ggtttttccc attactgaca tgtatactga gtcattaggg actttccaat
     2461 gggttttgcc cagtacataa ggtcaatagg ggtgaatcaa caggaaagtc ccattggagc
     2521 caagtacact gagtcaatag ggactttcca ttgggttttg cccagtacaa aaggtcaata
     2581 gggggtgagt caatgggttt ttcccattat tggcacatac ataaggtcaa taggggtgac
     2641 tagtggagaa gagcatgctt gagggctgag tgcccctcag tgggcagaga gcacatggcc
     2701 cacagtccct gagaagttgg ggggaggggt gggcaattga actggtgcct agagaaggtg
     2761 gggcttgggt aaactgggaa agtgatgtgg tgtactggct ccaccttttt ccccagggtg
     2821 ggggagaacc atatataagt gcagtagtct ctgtgaacat tcaagcttct gccttctccc
     2881 tcctgtgagt ttggtaagtc actgactgtc tatgcctggg aaagggtggg caggaggtgg
     2941 ggcagtgcag gaaaagtggc actgtgaacc ctgcagccct agacaattgt actaaccttc
     3001 ttctctttcc tctcctgaca ggttggtgta cagtagcttc cagaattcgc aaacatggtg
     3061 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     3121 gtaaacggcc acaagttcag cgtccgcggc gagggcgagg gcgatgccac caacggcaag
     3181 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     3241 accaccttcg gctacggcgt ggcctgcttc agccgctacc ccgaccacat gaagcagcac
     3301 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat ctctttcaag
     3361 gacgacggca cctacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     3421 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     3481 gagtacaact tcaacagcca ctacgtctat atcacggccg acaagcagaa gaactgcatc
     3541 aaggctaact tcaagatccg ccacaacgtt gaggacggca gcgtgcagct cgccgaccac
     3601 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     3661 agccatcagt ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     3721 gagttcgtga ccgccgccgg gattacacat ggcatggacg agctgtacaa gtaaatctag
     3781 aactatagtg agtcgtatta cgtagatcca gacatgataa gatacattga tgagtttgga
     3841 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     3901 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     3961 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aattcgcggc cgccaccgcg
     4021 gtggagctcc agcttttgtt ccctttagtg agggttaatt gggcccaagt gatctccaaa
     4081 aaataagtac tttttgactg taaataaaat tgtaaggagt aaaaagtact tttttttcta
     4141 aaaaaatgta attaagtaaa agtaaaagta ttgattttta attgtactca agtaaagtaa
     4201 aaatccccaa aaataatact taagtacagt aatcaagtaa aattactcaa gtactttaca
     4261 cctctgggta ctgtttcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4321 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4381 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4441 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4501 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4561 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4621 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4681 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4741 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4801 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4861 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4921 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4981 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5041 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5101 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5161 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5221 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5281 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5341 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5401 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5461 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5521 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5581 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5641 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5701 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5761 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5821 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5881 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5941 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6001 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6061 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6121 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6181 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6241 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6301 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6361 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6421 cgcacatttc cccgaaaagt gccac
//
