LOCUS       sequence-240979-        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"
     primer_bind     complement(110..131)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     322..341
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     554..576
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     568..585
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     569..585
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     592..611
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        592..610
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      641..650
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      645..654
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             663..683
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1725..1778
                     /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             1785..2495
                     /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     2198..2217
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2547..2668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2558..2577)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2612..2631
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2792..2839)
                     /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        2933..3037
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        3039..3229
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3062..3082)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             3269..4063
                     /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             3269..4063
                     /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(3323..3342)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3933..3952
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4070..4118
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    complement(4143..4190)
                     /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(4319..4337)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4320..4337)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4355..4371)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4355..4371)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4368..4390)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4379..4395
                     /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(4403..4409,4410..4427,4428..4433))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4448..4469
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4586..4603)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4757..5345)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4837..4856)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5516..6307,6308..6376))
                     /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     6139..6158
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6377..6481)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(6559..6586,1..428))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
       61 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
      121 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
      181 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
      241 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
      301 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
      361 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
      421 gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
      481 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
      541 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac
      601 tcactatagg gcgaattggg cccgacgtcg catgctcccg gccgccatgg cggccgcgga
      661 tgcccaagaa aaagcgcaaa gtgtccaacc tgttgacagt gcatcagaac ctgcctgccc
      721 tgcctgtgga cgcgacaagc gacgaggtcc gcaagaatct tatggacatg tttcgggacc
      781 gacaggcatt ctctgagcac acttggaaaa tgctgctgtc cgtgtgccgt tcttgggctg
      841 cctggtgtaa gctgaacaac agaaagtggt tcccggctga accggaagat gttcgtgact
      901 atctgcttta cctccaagct agaggtttgg ccgtcaagac aatccagcag catctggggc
      961 agcttaacat gctgcatcgg cgaagtggtc tgccaaggcc aagtgattca aacgctgtgt
     1021 ctctcgtgat gcgaagaatt aggaaagaga acgtggacgc tggagagaga gcgaagcagg
     1081 ctttggcttt cgagcgtact gatttcgacc aagtgcgctc tttgatggag aatagtgata
     1141 ggtgtcagga tataagaaat ttggccttcc tgggtattgc atataacaca cttttgcgca
     1201 ttgctgagat cgcccgcata cgggttaaag acatctctag aaccgacggg ggaaggatgc
     1261 tcatccacat cggcaggacg aaaaccctcg tgtccaccgc cggcgtcgag aaggcactgt
     1321 ctctgggagt gacaaaactt gtggagcgtt ggatcagtgt atcaggtgta gccgatgatc
     1381 ctaataacta ccttttctgc cgggtcagga agaacggcgt cgctgcccct tctgccacca
     1441 gtcagctgtc cacgcgcgcg ctggaaggaa tctttgaagc gacgcataga ctcatttatg
     1501 gagcgaaaga cgactctggc cagcgttatc tggcctggag tggacactct gctcgcgtgg
     1561 gagcggcgcg cgatatggca agagctggag tttctatccc tgagatcatg caggcaggtg
     1621 gatggacaaa cgttaacatc gtgatgaatt atattcgtaa cctggatagc gagaccggag
     1681 cgatggttcg gctgctcgag gatggagacg atatcggatc aggagagggc aggggaagtc
     1741 ttctaacatg cggggacgtg gaggaaaatc ccgggcccgg aagtatggtg tcgaagggcg
     1801 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
     1861 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     1921 cccagaccgc caagcttaaa gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     1981 tgtcccctca attcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     2041 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     2101 acggcggcgt ggtgaccgtg acccaggact cctccctcca agatggcgag ttcatttata
     2161 aagtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
     2221 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
     2281 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     2341 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
     2401 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     2461 actccaccgg cggcatggac gagctgtaca agtaaagcgg ccgcgactct agagtcgggg
     2521 cggccggccg cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac
     2581 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     2641 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttcg
     2701 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaa
     2761 aatcgataag gatccgtcga ccgacctgct tgaagttcct atactttcta gagaatagga
     2821 acttcggaat aggaacttca agatccccct ggcgaaaggg ggatgtgctg caaggcgatt
     2881 aagttgggta acgccagggt tttcgtcagg tggcactttt cggggaaatg tgcgcggaac
     2941 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3001 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     3061 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3121 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3181 ggcagaagta tgcaaagcat gcatctcaat tagtcagtaa ccatagtccg caaagatcga
     3241 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     3301 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     3361 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     3421 cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc
     3481 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     3541 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     3601 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     3661 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     3721 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     3781 cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     3841 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     3901 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     3961 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     4021 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgaggggatc ggcaataaaa
     4081 agacagaata aaacgcacgg gtgttgggtc gtttgttcgg atccgagctt caaaagcgct
     4141 ctgaagttcc tatactttct agagaatagg aacttcggaa tagtaacttc tccatggtag
     4201 cctccaaaaa agcctcctca ctacttctgg aatagctcaa tcactagtga attcgcggcc
     4261 gcctgcaggt cgaccatatg ggagagctcc caacgcgttg gatgcatagc ttgagtattc
     4321 tatagtgtca cctaaatagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4381 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4441 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4501 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4561 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4621 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4681 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4741 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4801 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4861 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4921 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4981 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5041 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5101 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5161 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5221 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5281 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5341 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5401 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5461 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5521 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5581 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5641 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5701 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5761 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5821 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5881 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5941 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6001 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6061 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6121 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6181 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6241 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6301 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6361 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6421 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6481 gcacatttcc ccgaaaagtg ccacctgatg cggtgtgaaa taccgcacag atgcgtaagg
     6541 agaaaatacc gcatcaggaa attgtaagcg ttaatatttt gttaaa
//
