LOCUS       sequence-492090-        6603 bp    DNA     circular SYN 06-NOV-2025
DEFINITION  AAV plasmid for expressing taCaspase-3 and T2A linked TEVp for 
            caspase mediated cell ablation, and an intersectional strategy for 
            Cre-off and Flp-on expression. See comments for more detail..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6603)
  TITLE     Coff/Fon Caspase3
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6603)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-Ef1a-Con/Fon-taCasp3-T2A-TEVp-WPRE3-BGHpA
FEATURES             Location/Qualifiers
     source          1..6603
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     protein_bind    137..157
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        158..1336
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          389..1327
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1284..1304
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1336..1360
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1361..1394)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1413..1446
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(2244..2297)
                     /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             complement(2613..2633)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     regulatory      3152..3161
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3168..3201)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(3298..3331)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(3332..3356)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3417..3437)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(3634..3651)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3640..3847
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3855..3995
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3855..3984
                     /label=AAV2 ITR
     rep_origin      4070..4525
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4157..4176)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4367..4388
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4542..4561)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4661..4683
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4721..4739)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4807..4911
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4912..4980,4981..5772)
                     /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     complement(5130..5149)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5943..6531
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6432..6451
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6593..6603,1..130)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tctagacaac tttgtataga aaagttgggc tccggtgccc gtcagtgggc
      181 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg
      241 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
      301 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
      361 tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg
      421 cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacctggc tgcagtacgt
      481 gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta
      541 aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt
      601 gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta
      661 aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg
      721 ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc
      781 gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg
      841 ggggtagtct caagctggcc ggcctgctct ggtgcctggt ctcgcgccgc cgtgtatcgc
      901 cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc
      961 gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc
     1021 gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga
     1081 ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac
     1141 gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt
     1201 ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt
     1261 tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc
     1321 atttcaggtg tcgtgacaag tttgtacaaa aaagcaggct ataacttcgt atagcataca
     1381 ttatacgaag ttatcctagg gtcgactccg gagaagttcc tattctctag aaagtatagg
     1441 aacttcgcag aatggtagct ggattgtagc tgctattagc aatatgaaac ctcttagaag
     1501 ttcctattct tcaaaaggta taggaacttc ggcgcgcctc aaagctgagt ggcttcctta
     1561 acgggctgga agggttcctc gggcttgacc atgaacacct tatgtccgcc ccaaagaacg
     1621 ctgtcggcgt tcagcctcca gccgctcacc cactgctgcg cctcttgatt ggtcaacagc
     1681 tccatgaagt ttttcggcac gctcgtgaag tagttgttgg tattggtaaa attgctggcg
     1741 ctgtggatgc ccacgatgaa gccgtcccta gtgctcacca atgggctgcc gcattggcca
     1801 tccttcgtct gaatccagtg cttccagaag atgccgtcgc tgctggggaa ggtgcagctg
     1861 gtatcagaga ccatgctgct catggacttg gtctggaagt tggtggtcac caggcagatt
     1921 cgctcctccc tttgtggctc cctgaacttc agtttctggg gaaagggggg gaagtccttg
     1981 ggcatcctga tgataatcat gtccctgccg tcgatcaggt gctgttgcag ggtggttgtg
     2041 ttcttcactt tgaacacgcc gtgcaggctt tgcaccagca gtgtgccgtt attcctcctg
     2101 aacagatgct tgttggtgat gatgaagggg ccgaagccga tgccgtacag actagtggtg
     2161 tgaccgtcgc tctcgttggt caggtgacag atggtgctgg agatcgggtt gtagtccctc
     2221 gggcccttga aaaggctctc gccagggccg ggattctcct ccacgtcacc gcatgttaga
     2281 agacttcctc tgccctctcc gcttccggag tgataaaaat agagttcttt tgtgagcatg
     2341 gaaacaatac atggaatctg tttctttgca tgaaaagtag cgtcaaagga aaaggactca
     2401 aattctgttg ccacctttcg gttaacccgg gtaagaatgt gcataaattc aagcttgtcg
     2461 gcatactgtt tcagcatggc acaaagcgac tggatgaacc aggagccatc ctttgaattt
     2521 cgccaagaat aataaccagg tgctgtggag tatgcataca agaagtcggc ctccactggt
     2581 attttatgac acgccatgtc atcatcaaca ccactctgga agtagagatt ttctgtctca
     2641 atgccacagt ccagttctgt accacggcag gcctgaataa tgaaaagttt gggttttcca
     2701 gttagacttc tacaacgatc ccctctgaaa aagtttgtta tttttttcag gtcaacaggt
     2761 ccatttgttc caaaaattat tccttcttca ccatggctca gaagcacaca aacaaaactg
     2821 ctccttttgc tgtgatcttc tttagaaaca tcacgcatca attccacaat ttcttcacgt
     2881 gtaagatcat ttttattcct gacttcatat ttcaagtttc tgaatgtttc cctgaggttt
     2941 gctgcatcga catctgtacc agaccgagat gtcattccag tgcttttatg aaaattctta
     3001 ttattaatta ttatacataa acccatctca ggataatcca ttttataact gttgtccagg
     3061 gatattccag agtccattga ttcgcttcca tgtatgatct ttggttccaa atttttaatg
     3121 gattttgaat ccactgagtt ttcagtgttc tccatggtgg cgctagcgaa gttcctatac
     3181 tttctagaga ataggaactt cttgccttaa cccagaaatt atcactgtta ttctttagaa
     3241 tggtgcaaag agaagttcct ataccttttg aagaatagga acttcgaatt ctctagaata
     3301 acttcgtata gcatacatta tacgaagtta tacccagctt tcttgtacaa agtgggaatt
     3361 cataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     3421 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     3481 cgtatggctt tcattttctc ctccttgtat aaatcctggt tagttcttgc cacggcggaa
     3541 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3601 tccgtggtgg aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     3661 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3721 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3781 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg
     3841 ctggggaggg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     3901 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     3961 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     4021 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     4081 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4141 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4201 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4261 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     4321 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4381 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     4441 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4501 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     4561 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4621 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4681 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     4741 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4801 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4861 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4921 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4981 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5041 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5101 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5161 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5221 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5281 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5341 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5401 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5461 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5521 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5581 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc
     5641 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5701 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5761 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5821 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5881 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5941 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6001 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6061 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6121 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6181 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6241 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6301 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6361 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6421 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6481 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6541 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gtcctgcagg
     6601 cag
//
