LOCUS       sequence-239420-        5697 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor cassette containing myr-tagged eGFP 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-eGFP-SV40pA-FKF
FEATURES             Location/Qualifiers
     source          1..5697
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..27
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     19..36
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     20..36
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     43..62
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        43..61
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      92..101
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      96..105
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             120..146
                     /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             153..179
                     /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             186..212
                     /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      272..281
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(341..343,344..1057)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(383..404)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(644..663)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     991..1012
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1109..1230
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1120..1139)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1174..1193
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(1354..1401)
                     /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        1495..1599
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1601..1791
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1624..1644)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             1831..2625
                     /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             1831..2625
                     /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(1885..1904)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2495..2514
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2632..2680
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    complement(2705..2752)
                     /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(2881..2899)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2882..2899)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2917..2933)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2917..2933)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2930..2952)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2941..2957
                     /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(2965..2971,2972..2989,2990..2995))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3010..3031
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3148..3165)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3319..3907)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3399..3418)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4078..4869,4870..4938))
                     /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     4701..4720
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4939..5043)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5121..5576)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5258..5279)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5470..5489
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag
       61 ggcgaattgg gcccgacgtc gcatgctccc ggccgccatg gcggccgcgg atggccacct
      121 atgacgtccc agattacgca agtttgcccg gatatgatgt tcctgattat gctagcctcc
      181 cgggttacga tgtgcccgac tatgcctcac ttccaggcag atcatgggct gcatcaagag
      241 caagcgcaag gacaacctga acgacgacga ggccgccatg ggctgcatca agagcaagcg
      301 caaggacaac ctgaacgacg acgagggtgg aagcggatct gtgagcaagg gcgaggagct
      361 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
      421 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
      481 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
      541 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
      601 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
      661 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
      721 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
      781 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
      841 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
      901 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc agtccgccct
      961 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1021 cgggatcact ctcggcatgg acgagctgta caagtaaagc ggccgcgact ctagagtcgg
     1081 ggcggccggc cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca
     1141 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     1201 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     1261 cgggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     1321 aaaatcgata aggatccgtc gaccgacctg cttgaagttc ctatactttc tagagaatag
     1381 gaacttcgga ataggaactt caagatcccc ctggcgaaag ggggatgtgc tgcaaggcga
     1441 ttaagttggg taacgccagg gttttcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     1501 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     1561 ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc
     1621 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     1681 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     1741 caggcagaag tatgcaaagc atgcatctca attagtcagt aaccatagtc cgcaaagatc
     1801 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     1861 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     1921 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     1981 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg
     2041 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     2101 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     2161 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     2221 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     2281 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     2341 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     2401 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     2461 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     2521 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     2581 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagggga tcggcaataa
     2641 aaagacagaa taaaacgcac gggtgttggg tcgtttgttc ggatccgagc ttcaaaagcg
     2701 ctctgaagtt cctatacttt ctagagaata ggaacttcgg aatagtaact tctccatggt
     2761 agcctccaaa aaagcctcct cactacttct ggaatagctc aatcactagt gaattcgcgg
     2821 ccgcctgcag gtcgaccata tgggagagct cccaacgcgt tggatgcata gcttgagtat
     2881 tctatagtgt cacctaaata gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     2941 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3001 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3061 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3121 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3181 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3241 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3301 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3361 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3421 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3481 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3541 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3601 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3661 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3721 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     3781 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3841 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3901 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3961 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4021 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4081 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4141 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4201 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4261 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4321 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4381 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4441 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4501 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4561 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4621 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4681 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4741 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4801 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4861 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4921 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4981 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5041 gcgcacattt ccccgaaaag tgccacctga tgcggtgtga aataccgcac agatgcgtaa
     5101 ggagaaaata ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     5161 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5221 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5281 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     5341 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5401 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     5461 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     5521 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat
     5581 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     5641 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccaggg
//
