LOCUS       sequence-227054-        6586 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor cassette containing NLS-tagged Cre, a Thosea asigna virus 
            peptide (2A), mCherry 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 6586)
  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 6586)
  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-NLS-Cre-2A-mCherry-FKF
FEATURES             Location/Qualifiers
     source          1..6586
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(28..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(138..159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     350..369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     582..604
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     596..613
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     597..613
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     620..639
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        620..638
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      669..678
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      673..682
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             691..711
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1753..1806
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1813..2523
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     2226..2245
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2575..2696
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2586..2605)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2640..2659
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2820..2867)
                     /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        2961..3065
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        3067..3257
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3090..3110)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             3297..4091
                     /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             3297..4091
                     /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(3351..3370)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3961..3980
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4098..4146
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    complement(4171..4218)
                     /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(4347..4365)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4348..4365)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4383..4399)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4383..4399)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4396..4418)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4407..4423
                     /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(4431..4437,4438..4455,4456..4461))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4476..4497
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4614..4631)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4785..5373)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4865..4884)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5544..6335,6336..6404))
                     /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     6167..6186
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6405..6509)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
      121 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa
      481 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
      541 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
      601 aacgacggcc agtgaattgt aatacgactc actatagggc gaattgggcc cgacgtcgca
      661 tgctcccggc cgccatggcg gccgcggatg cccaagaaaa agcgcaaagt gtccaacctg
      721 ttgacagtgc atcagaacct gcctgccctg cctgtggacg cgacaagcga cgaggtccgc
      781 aagaatctta tggacatgtt tcgggaccga caggcattct ctgagcacac ttggaaaatg
      841 ctgctgtccg tgtgccgttc ttgggctgcc tggtgtaagc tgaacaacag aaagtggttc
      901 ccggctgaac cggaagatgt tcgtgactat ctgctttacc tccaagctag aggtttggcc
      961 gtcaagacaa tccagcagca tctggggcag cttaacatgc tgcatcggcg aagtggtctg
     1021 ccaaggccaa gtgattcaaa cgctgtgtct ctcgtgatgc gaagaattag gaaagagaac
     1081 gtggacgctg gagagagagc gaagcaggct ttggctttcg agcgtactga tttcgaccaa
     1141 gtgcgctctt tgatggagaa tagtgatagg tgtcaggata taagaaattt ggccttcctg
     1201 ggtattgcat ataacacact tttgcgcatt gctgagatcg cccgcatacg ggttaaagac
     1261 atctctagaa ccgacggggg aaggatgctc atccacatcg gcaggacgaa aaccctcgtg
     1321 tccaccgccg gcgtcgagaa ggcactgtct ctgggagtga caaaacttgt ggagcgttgg
     1381 atcagtgtat caggtgtagc cgatgatcct aataactacc ttttctgccg ggtcaggaag
     1441 aacggcgtcg ctgccccttc tgccaccagt cagctgtcca cgcgcgcgct ggaaggaatc
     1501 tttgaagcga cgcatagact catttatgga gcgaaagacg actctggcca gcgttatctg
     1561 gcctggagtg gacactctgc tcgcgtggga gcggcgcgcg atatggcaag agctggagtt
     1621 tctatccctg agatcatgca ggcaggtgga tggacaaacg ttaacatcgt gatgaattat
     1681 attcgtaacc tggatagcga gaccggagcg atggttcggc tgctcgagga tggagacgat
     1741 atcggatcag gagagggcag gggaagtctt ctaacatgcg gggacgtgga ggaaaatccc
     1801 gggcccggaa gtatggtgtc gaagggcgag gaggataaca tggccatcat caaggagttc
     1861 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
     1921 gagggcgagg gccgccccta cgagggcacc cagaccgcca agcttaaagt gaccaagggt
     1981 ggccccctgc ccttcgcctg ggacatcctg tcccctcaat tcatgtacgg ctccaaggcc
     2041 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
     2101 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
     2161 tccctccaag atggcgagtt catttataaa gtgaagctgc gcggcaccaa cttcccctcc
     2221 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     2281 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     2341 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     2401 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     2461 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     2521 taaagcggcc gcgactctag agtcggggcg gccggccgct tcgagcagac atgataagat
     2581 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     2641 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     2701 acaacaattg cattcatttt atgtttcggg ttcaggggga ggtgtgggag gttttttaaa
     2761 gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tccgtcgacc gacctgcttg
     2821 aagttcctat actttctaga gaataggaac ttcggaatag gaacttcaag atccccctgg
     2881 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcgtcaggtg
     2941 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3001 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3061 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3121 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3181 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3241 gtcagtaacc atagtccgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     3301 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3361 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3421 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     3481 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3541 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3601 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3661 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3721 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3781 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     3841 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3901 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3961 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     4021 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4081 agttcttctg aggggatcgg caataaaaag acagaataaa acgcacgggt gttgggtcgt
     4141 ttgttcggat ccgagcttca aaagcgctct gaagttccta tactttctag agaataggaa
     4201 cttcggaata gtaacttctc catggtagcc tccaaaaaag cctcctcact acttctggaa
     4261 tagctcaatc actagtgaat tcgcggccgc ctgcaggtcg accatatggg agagctccca
     4321 acgcgttgga tgcatagctt gagtattcta tagtgtcacc taaatagctt ggcgtaatca
     4381 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4441 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4501 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4561 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4621 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4681 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4741 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4801 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4861 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4921 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4981 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5041 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5101 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5161 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5221 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5281 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5341 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5401 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5461 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5521 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5581 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5641 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5701 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5761 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5821 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5881 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5941 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6001 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6061 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6121 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6181 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6241 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6301 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6361 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6421 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6481 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgatgcg
     6541 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagg
//
