LOCUS       sequence-239419-        5697 bp    DNA     circular SYN 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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
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="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     86..108
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     100..117
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     101..117
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     124..143
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        124..142
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      173..182
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      177..186
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             201..227
                     /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             234..260
                     /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             267..293
                     /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      353..362
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(422..424,425..1135)
                     /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(464..485)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(725..744)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1072..1093
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1190..1311
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1201..1220)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1255..1274
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(1435..1482)
                     /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        1576..1680
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1682..1872
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1705..1725)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             1912..2706
                     /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             1912..2706
                     /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(1966..1985)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2576..2595
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2713..2761
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    complement(2786..2833)
                     /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(2962..2980)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2963..2980)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2998..3014)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2998..3014)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3011..3033)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3022..3038
                     /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(3046..3052,3053..3070,3071..3076))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3091..3112
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3229..3246)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3400..3988)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3480..3499)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4159..4950,4951..5019))
                     /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     4782..4801
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5020..5124)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5202..5657)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5339..5360)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5551..5570
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
       61 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa
      121 ttgtaatacg actcactata gggcgaattg ggcccgacgt cgcatgctcc cggccgccat
      181 ggcggccgcg gatggccacc tatgacgtcc cagattacgc aagtttgccc ggatatgatg
      241 ttcctgatta tgctagcctc ccgggttacg atgtgcccga ctatgcctca cttccaggca
      301 gatcatgggc tgcatcaaga gcaagcgcaa ggacaacctg aacgacgacg aggccgccat
      361 gggctgcatc aagagcaagc gcaaggacaa cctgaacgac gacgagggtg gaagcggatc
      421 tgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
      481 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
      541 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
      601 cgtgaccacc ttcggctacg gcctgatgtg cttcgcccgc taccccgacc acatgaagca
      661 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
      721 caaagacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
      781 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
      841 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
      901 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
      961 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     1021 cctgagctac cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1081 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag
     1141 cggccgcgac tctagagtcg gggcggccgg ccgcttcgag cagacatgat aagatacatt
     1201 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     1261 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     1321 aattgcattc attttatgtt tcgggttcag ggggaggtgt gggaggtttt ttaaagcaag
     1381 taaaacctct acaaatgtgg taaaatcgat aaggatccgt cgaccgacct gcttgaagtt
     1441 cctatacttt ctagagaata ggaacttcgg aataggaact tcaagatccc cctggcgaaa
     1501 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttcgtc aggtggcact
     1561 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1621 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     1681 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     1741 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     1801 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     1861 taaccatagt ccgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     1921 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     1981 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     2041 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     2101 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     2161 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     2221 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     2281 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     2341 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     2401 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     2461 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     2521 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     2581 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     2641 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     2701 ttctgagggg atcggcaata aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt
     2761 cggatccgag cttcaaaagc gctctgaagt tcctatactt tctagagaat aggaacttcg
     2821 gaatagtaac ttctccatgg tagcctccaa aaaagcctcc tcactacttc tggaatagct
     2881 caatcactag tgaattcgcg gccgcctgca ggtcgaccat atgggagagc tcccaacgcg
     2941 ttggatgcat agcttgagta ttctatagtg tcacctaaat agcttggcgt aatcatggtc
     3001 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3061 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3121 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3181 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3241 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3301 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3361 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3421 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3481 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3541 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3601 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3661 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3721 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3781 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3841 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3901 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3961 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4021 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4081 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4141 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4201 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4261 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4321 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4381 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4441 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4501 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4561 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4621 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4681 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4741 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4801 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4861 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4921 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4981 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5041 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5101 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg atgcggtgtg
     5161 aaataccgca cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat
     5221 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     5281 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     5341 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     5401 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     5461 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     5521 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     5581 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     5641 gccgctacag ggcgcgtcca ttcgccattc aggctgcgca actgttggga agggcga
//
