LOCUS       sequence-386831-        6682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian/zebrafish expression of zemiRFP2-EF1-2xCMV-EF1-mClover3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6682)
  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 6682)
  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: pTol2zemiRFP2-EF1-2xCMV-EF1-mClover3
FEATURES             Location/Qualifiers
     source          1..6682
                     /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(3292..3294,3295..3297,3298..4011)
                     /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(3337..3358)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3945..3966
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4042..4176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(4279..4297)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    join(4304..4491,4492..4503)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4533..4549)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4533..4549)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4546..4568)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4557..4573
                     /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(4581..4587,4588..4605,4606..4611))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4626..4647
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4764..4781)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4935..5523)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5015..5034)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5694..6485,6486..6554))
                     /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     6317..6336
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6555..6659)
                     /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 tcacatgtca ggaggtcagg
     1441 ctgtctagcc acgcttccct cggccatggt ggatatcgtt taaacaattc tggaagctac
     1501 tgtacaccaa cctgtcagga gaggaaagag aagaaggtta gtacaattgt ctagggctgc
     1561 agggttcaca gtgccacttt tcctgcactg ccccacctcc tgcccaccct ttcccaggca
     1621 tagacagtca gtgacttacc aaactcacag gagggagaag gcagaagctt gaatgttcac
     1681 agagactact gcacttatat atggttctcc cccaccctgg ggaaaaaggt ggagccagta
     1741 caccacatca ctttcccagt ttacccaagc cccaccttct ctaggcacca gttcaattgc
     1801 ccacccctcc ccccaacttc tcagggactg tgggccatgt gctctctgcc cactgagggg
     1861 cactcagccc tcaagcatgc tcttctccac tagtcacccc tattgacctt atgtatgtgc
     1921 caataatggg aaaaacccat tgactcaccc cctattgacc ttttgtactg ggcaaaaccc
     1981 aatggaaagt ccctattgac tcagtgtact tggctccaat gggactttcc tgttgattca
     2041 cccctattga ccttatgtac tgggcaaaac ccattggaaa gtccctaatg actcagtata
     2101 catgtcagta atgggaaaaa cccattggct tacctcccat tgaccttatg tactgggcaa
     2161 aacccattgg aaagtcccta ttgactcaat gtacttggct ccaatgggac tttcctgttg
     2221 actcaccccc tattgacctt atgtactggg caaaacccaa tggaaagtcc ctattgagtc
     2281 agtgtacttg gctccaatgg gtttttccca ttgactcctg cattaatcga tttaaatgct
     2341 agggctgctc tccaatctct gtcatggcag ggccactggc atccctgaag ttggtggaga
     2401 ccacctcact ccactcagca tacagctcat ccagtcctct taattaatgc aggagtcaat
     2461 gggaaaaacc cattggagcc aagtacactg actcaatagg gactttccat tgggttttgc
     2521 ccagtacata aggtcaatag ggggtgagtc aacaggaaag tcccattgga gccaagtaca
     2581 ttgagtcaat agggactttc caatgggttt tgcccagtac ataaggtcaa tgggaggtaa
     2641 gccaatgggt ttttcccatt actgacatgt atactgagtc attagggact ttccaatggg
     2701 ttttgcccag tacataaggt caataggggt gaatcaacag gaaagtccca ttggagccaa
     2761 gtacactgag tcaataggga ctttccattg ggttttgccc agtacaaaag gtcaataggg
     2821 ggtgagtcaa tgggtttttc ccattattgg cacatacata aggtcaatag gggtgactag
     2881 tggagaagag catgcttgag ggctgagtgc ccctcagtgg gcagagagca catggcccac
     2941 agtccctgag aagttggggg gaggggtggg caattgaact ggtgcctaga gaaggtgggg
     3001 cttgggtaaa ctgggaaagt gatgtggtgt actggctcca cctttttccc cagggtgggg
     3061 gagaaccata tataagtgca gtagtctctg tgaacattca agcttctgcc ttctccctcc
     3121 tgtgagtttg gtaagtcact gactgtctat gcctgggaaa gggtgggcag gaggtggggc
     3181 agtgcaggaa aagtggcact gtgaaccctg cagccctaga caattgtact aaccttcttc
     3241 tctttcctct cctgacaggt tggtgtacag tagcttccag aattcgcaaa catggtgagc
     3301 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     3361 aacggccaca agttcagcgt ccgcggcgag ggcgagggcg atgccaccaa cggcaagctg
     3421 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     3481 accttcggct acggcgtggc ctgcttcagc cgctaccccg accacatgaa gcagcacgac
     3541 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctc tttcaaggac
     3601 gacggcacct acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     3661 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     3721 tacaacttca acagccacta cgtctatatc acggccgaca agcagaagaa ctgcatcaag
     3781 gctaacttca agatccgcca caacgttgag gacggcagcg tgcagctcgc cgaccactac
     3841 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     3901 catcagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     3961 ttcgtgaccg ccgccgggat tacacatggc atggacgagc tgtacaagta aatctagaac
     4021 tatagtgagt cgtattacgt agatccagac atgataagat acattgatga gtttggacaa
     4081 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     4141 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     4201 atgtttcagg ttcaggggga ggtgtgggag gttttttaat tcgcggccgc caccgcggtg
     4261 gagctccagc ttttgttccc tttagtgagg gttaattggg cccaagtgat ctccaaaaaa
     4321 taagtacttt ttgactgtaa ataaaattgt aaggagtaaa aagtactttt ttttctaaaa
     4381 aaatgtaatt aagtaaaagt aaaagtattg atttttaatt gtactcaagt aaagtaaaaa
     4441 tccccaaaaa taatacttaa gtacagtaat caagtaaaat tactcaagta ctttacacct
     4501 ctgggtactg tttcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4561 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4621 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4681 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4741 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4801 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4861 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4921 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4981 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5041 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5101 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5161 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5221 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5281 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5341 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5401 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5461 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5521 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5581 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5641 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5701 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5761 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5821 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5881 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5941 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6001 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6061 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6121 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6181 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6241 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6301 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6361 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6421 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6481 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6541 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6601 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6661 acatttcccc gaaaagtgcc ac
//
