LOCUS       sequence-227057-        5697 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Donor cassette containing myr-tagged Citrine and SV40 polyA tail, 
            followed by FRT site-flanked Kanamycin selection gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5697)
  AUTHORS   Weinberger M, Simoes FC, Patient R, Sauka-Spengler T, Riley PR
  TITLE     Functional Heterogeneity within the Developing Zebrafish Epicardium.
  JOURNAL   Dev Cell. 2020 Mar 9;52(5):574-590.e6. doi: 
            10.1016/j.devcel.2020.01.023. Epub 2020 Feb 20.
  PUBMED    32084358
REFERENCE   2  (bases 1 to 5697)
  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: 
            "10.1016/j.devcel.2020.01.023"; journalName: "Dev Cell"; date: 
            "2020-03-9- 9"; volume: "52"; issue: "5"; pages: "574-590.e6"
COMMENT     Sequence Label: pGEM-myr-Citrine-SV40pA-FKF
FEATURES             Location/Qualifiers
     source          1..5697
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     12..31
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        12..30
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      61..70
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      65..74
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             89..115
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             122..148
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             155..181
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     regulatory      241..250
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(310..312,313..1023)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(352..373)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(613..632)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     960..981
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1078..1199
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1089..1108)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1143..1162
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(1323..1370)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        1464..1568
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1570..1760
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1593..1613)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             1800..2594
                     /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             1800..2594
                     /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(1854..1873)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2464..2483
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2601..2649
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    complement(2674..2721)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        complement(2850..2868)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2851..2868)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2886..2902)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2886..2902)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2899..2921)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2910..2926
                     /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(2934..2940,2941..2958,2959..2964))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2979..3000
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3117..3134)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3288..3876)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3368..3387)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4047..4838,4839..4907))
                     /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     4670..4689
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4908..5012)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5090..5545)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5227..5248)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5439..5458
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5671..5693
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ccagtgaatt gtaatacgac tcactatagg gcgaattggg cccgacgtcg catgctcccg
       61 gccgccatgg cggccgcgga tggccaccta tgacgtccca gattacgcaa gtttgcccgg
      121 atatgatgtt cctgattatg ctagcctccc gggttacgat gtgcccgact atgcctcact
      181 tccaggcaga tcatgggctg catcaagagc aagcgcaagg acaacctgaa cgacgacgag
      241 gccgccatgg gctgcatcaa gagcaagcgc aaggacaacc tgaacgacga cgagggtgga
      301 agcggatctg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      361 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      421 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
      481 cccaccctcg tgaccacctt cggctacggc ctgatgtgct tcgcccgcta ccccgaccac
      541 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
      601 atcttcttca aagacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
      661 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
      721 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
      781 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
      841 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
      901 aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
      961 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1021 aagtaaagcg gccgcgactc tagagtcggg gcggccggcc gcttcgagca gacatgataa
     1081 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     1141 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     1201 acaacaacaa ttgcattcat tttatgtttc gggttcaggg ggaggtgtgg gaggtttttt
     1261 aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatccgtcg accgacctgc
     1321 ttgaagttcc tatactttct agagaatagg aacttcggaa taggaacttc aagatccccc
     1381 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcgtcag
     1441 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     1501 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     1561 ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     1621 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     1681 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     1741 ttagtcagta accatagtcc gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     1801 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     1861 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     1921 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     1981 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     2041 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     2101 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     2161 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     2221 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     2281 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     2341 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     2401 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     2461 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     2521 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     2581 acgagttctt ctgaggggat cggcaataaa aagacagaat aaaacgcacg ggtgttgggt
     2641 cgtttgttcg gatccgagct tcaaaagcgc tctgaagttc ctatactttc tagagaatag
     2701 gaacttcgga atagtaactt ctccatggta gcctccaaaa aagcctcctc actacttctg
     2761 gaatagctca atcactagtg aattcgcggc cgcctgcagg tcgaccatat gggagagctc
     2821 ccaacgcgtt ggatgcatag cttgagtatt ctatagtgtc acctaaatag cttggcgtaa
     2881 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     2941 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3001 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3061 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3121 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3181 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3241 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3301 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3361 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3421 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3481 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3541 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3601 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3661 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3721 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3781 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3841 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3901 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     3961 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4021 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4081 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4141 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4201 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4261 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4321 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4381 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4441 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4501 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4561 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4621 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4681 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4741 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4801 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4861 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4921 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4981 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgat
     5041 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     5101 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     5161 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     5221 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     5281 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     5341 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     5401 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     5461 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     5521 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     5581 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     5641 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacgg
//
