LOCUS       sequence-387242-        6697 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian/zebrafish expression of zmiRFP2-EF1-2xCMV-EF1-mClover3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6697)
  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 6697)
  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: pTol2zmiRFP2-EF1-2xCMV-EF1-mClover3
FEATURES             Location/Qualifiers
     source          1..6697
                     /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      1475..1484
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3307..3309,3310..3312,3313..4026)
                     /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(3352..3373)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3960..3981
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4057..4191
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(4294..4312)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    join(4319..4506,4507..4518)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4548..4564)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4548..4564)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4561..4583)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4572..4588
                     /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(4596..4602,4603..4620,4621..4626))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4641..4662
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4779..4796)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4950..5538)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5030..5049)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5709..6500,6501..6569))
                     /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     6332..6351
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6570..6674)
                     /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 ccactagttt agatatcgta
      541 acccagagct gtgattcttg tagcgatagc ctcagccagc agcacacaga tagctctcag
      601 ctcgaagtgg atgaatctag gcaggtagtg gtgacagatc accagtcccc acagctttcc
      661 tcccaccacc aggctcacca ccagtgtagc tctcactccc atgttcttca ggaactgcag
      721 gtgtgtaggg ctcatgcttc tcaggaaaca tccgctcatg tccaggtctc ttcctgtcag
      781 agggctcagt ctaggctcca gaggcacagg ctggtatgtc acgtccacca gcactctcac
      841 tctctgtctc acgtacagtc ttctagccat ctgaggcacc aggctgctag ggtatctgtt
      901 tccgaagtag ctctccagtc ctgtcacgtg gatctcgctg tacacctctc cgtgtcctct
      961 ctcgtcgaat ctgtacacca tcactctgtc gtatcctgta cactgctgga acagcagcac
     1021 tgtgtcgtca cacagagctc tcaggcttcc agctgttctg attctctcca gagcaggagc
     1081 cagtgttccg ctcaggtcga tgctaggtcc agctctctcc agctcgatga tcagtcctcc
     1141 ctcaggaggt ctgtgcatca gtccgtcgta ctctgtgcta gggtttccga ttctacatct
     1201 cacagccaca ggcattccct cagctgtcag gtccaggtga ggcaggatct tgatcagcag
     1261 gtctccgtcg atctcagcca gagggattcc cagcacgctt cccaggttca ggaactcagc
     1321 agcgttagcg ctagcctgga tgattctgtg gtcaggctcg ctcaccacca gcagtgttcc
     1381 gtgaggctgg atgcttccag ccaggtggat ctcctcgtgc tcacagtcgc tagggctagc
     1441 tgttccgaag tcagggcttc cgctagcgtg tccagccacc atggtggata tcgtttaaac
     1501 aattctggaa gctactgtac accaacctgt caggagagga aagagaagaa ggttagtaca
     1561 attgtctagg gctgcagggt tcacagtgcc acttttcctg cactgcccca cctcctgccc
     1621 accctttccc aggcatagac agtcagtgac ttaccaaact cacaggaggg agaaggcaga
     1681 agcttgaatg ttcacagaga ctactgcact tatatatggt tctcccccac cctggggaaa
     1741 aaggtggagc cagtacacca catcactttc ccagtttacc caagccccac cttctctagg
     1801 caccagttca attgcccacc cctcccccca acttctcagg gactgtgggc catgtgctct
     1861 ctgcccactg aggggcactc agccctcaag catgctcttc tccactagtc acccctattg
     1921 accttatgta tgtgccaata atgggaaaaa cccattgact caccccctat tgaccttttg
     1981 tactgggcaa aacccaatgg aaagtcccta ttgactcagt gtacttggct ccaatgggac
     2041 tttcctgttg attcacccct attgacctta tgtactgggc aaaacccatt ggaaagtccc
     2101 taatgactca gtatacatgt cagtaatggg aaaaacccat tggcttacct cccattgacc
     2161 ttatgtactg ggcaaaaccc attggaaagt ccctattgac tcaatgtact tggctccaat
     2221 gggactttcc tgttgactca ccccctattg accttatgta ctgggcaaaa cccaatggaa
     2281 agtccctatt gagtcagtgt acttggctcc aatgggtttt tcccattgac tcctgcatta
     2341 atcgatttaa atgctagggc tgctctccaa tctctgtcat ggcagggcca ctggcatccc
     2401 tgaagttggt ggagaccacc tcactccact cagcatacag ctcatccagt cctcttaatt
     2461 aatgcaggag tcaatgggaa aaacccattg gagccaagta cactgactca atagggactt
     2521 tccattgggt tttgcccagt acataaggtc aatagggggt gagtcaacag gaaagtccca
     2581 ttggagccaa gtacattgag tcaataggga ctttccaatg ggttttgccc agtacataag
     2641 gtcaatggga ggtaagccaa tgggtttttc ccattactga catgtatact gagtcattag
     2701 ggactttcca atgggttttg cccagtacat aaggtcaata ggggtgaatc aacaggaaag
     2761 tcccattgga gccaagtaca ctgagtcaat agggactttc cattgggttt tgcccagtac
     2821 aaaaggtcaa tagggggtga gtcaatgggt ttttcccatt attggcacat acataaggtc
     2881 aataggggtg actagtggag aagagcatgc ttgagggctg agtgcccctc agtgggcaga
     2941 gagcacatgg cccacagtcc ctgagaagtt ggggggaggg gtgggcaatt gaactggtgc
     3001 ctagagaagg tggggcttgg gtaaactggg aaagtgatgt ggtgtactgg ctccaccttt
     3061 ttccccaggg tgggggagaa ccatatataa gtgcagtagt ctctgtgaac attcaagctt
     3121 ctgccttctc cctcctgtga gtttggtaag tcactgactg tctatgcctg ggaaagggtg
     3181 ggcaggaggt ggggcagtgc aggaaaagtg gcactgtgaa ccctgcagcc ctagacaatt
     3241 gtactaacct tcttctcttt cctctcctga caggttggtg tacagtagct tccagaattc
     3301 gcaaacatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     3361 ctggacggcg acgtaaacgg ccacaagttc agcgtccgcg gcgagggcga gggcgatgcc
     3421 accaacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     3481 cccaccctcg tgaccacctt cggctacggc gtggcctgct tcagccgcta ccccgaccac
     3541 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     3601 atctctttca aggacgacgg cacctacaag acccgcgccg aggtgaagtt cgagggcgac
     3661 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     3721 gggcacaagc tggagtacaa cttcaacagc cactacgtct atatcacggc cgacaagcag
     3781 aagaactgca tcaaggctaa cttcaagatc cgccacaacg ttgaggacgg cagcgtgcag
     3841 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     3901 aaccactacc tgagccatca gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac
     3961 atggtcctgc tggagttcgt gaccgccgcc gggattacac atggcatgga cgagctgtac
     4021 aagtaaatct agaactatag tgagtcgtat tacgtagatc cagacatgat aagatacatt
     4081 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     4141 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     4201 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaattcgcg
     4261 gccgccaccg cggtggagct ccagcttttg ttccctttag tgagggttaa ttgggcccaa
     4321 gtgatctcca aaaaataagt actttttgac tgtaaataaa attgtaagga gtaaaaagta
     4381 cttttttttc taaaaaaatg taattaagta aaagtaaaag tattgatttt taattgtact
     4441 caagtaaagt aaaaatcccc aaaaataata cttaagtaca gtaatcaagt aaaattactc
     4501 aagtacttta cacctctggg tactgtttcg cgcttggcgt aatcatggtc atagctgttt
     4561 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4621 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4681 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4741 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4801 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4861 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4921 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4981 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5041 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5101 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5161 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5221 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5281 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5341 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5401 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5461 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5521 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5581 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5641 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5701 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5761 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5821 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5881 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5941 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6001 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6061 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6121 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6181 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6241 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6301 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6361 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6421 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6481 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6541 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6601 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6661 ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
