LOCUS       sequence-308249-        6500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for Flp-dependent expression of EGFP tagged KASH protein 
            domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6500)
  AUTHORS   Hunker AC, Soden ME, Krayushkina D, Heymann G, Awatramani R, Zweifel
            LS
  TITLE     Conditional Single Vector CRISPR/SaCas9 Viruses for Efficient 
            Mutagenesis in the Adult Mouse Nervous System.
  JOURNAL   Cell Rep. 2020 Mar 24;30(12):4303-4316.e6. doi: 
            10.1016/j.celrep.2020.02.092.
  PUBMED    32209486
REFERENCE   2  (bases 1 to 6500)
  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.celrep.2020.02"; journalName: "Cell Rep"; date: 
            "2020-03-24- 24"; volume: "30"; issue: "12"; pages: "4303-4316.e6"
COMMENT     Sequence Label: pAAV-FLEXfrt-EGFP-KASH
FEATURES             Location/Qualifiers
     source          1..6500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     primer_bind     957..979
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1045..1064
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1097..1130
                     /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(join(1448..2158,2159..2161,2162..2164))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1448..2158,2159..2161,2162..2164))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1490..1511)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1839..1858
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2098..2119
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    2320..2908
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2373..2393)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2791..2802)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2935..2952)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2941..3165
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(3375..3704)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3389..3524
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3689..3709
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3950..4538)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4030..4049)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4709..5500,5501..5569))
                     /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     5332..5351
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5570..5674)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5742..5760
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5798..5820)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5920..5939
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6153..6172
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6462..6480
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6462..6479
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 aggtaccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      241 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      301 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      361 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      421 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
      481 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
      541 cggggggggg gggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc
      601 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
      661 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc
      721 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg
      781 cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct
      841 tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc
      901 ggctcggggc tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt
      961 cggcttctgg cgtgtgaccg gcggctctag ctagagcctc tgctaaccat gttcatgcct
     1021 tcttcttttt cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg
     1081 gcaaagaatt ggatccgaag ttcctattct ctagaaagta taggaacttc caaggcctga
     1141 agttcctatt cttcaaaagg tataggaact tcgcggccgc ctaggtggga ggtggcccgt
     1201 tggtgtaccg cagcatgggg tagaaggatc gggcaaagtt gttggcctgg gtgcagctgt
     1261 agtcatcttc agaggcaggt agcaggcagg ccaggagcag cagcagcagc agaagcagct
     1321 gcaacggtag cgctgccctg atcacccttg agaggaagga gcgtctcggc tgggagctgc
     1381 cggggctgtc gaggtggggc atcctgctgt ctgtctcctc ctcctcctct cgagatctga
     1441 gtccggactt gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca
     1501 gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac tgggtgctca
     1561 ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt
     1621 ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga
     1681 tgccgttctt ctgcttgtcg gccatgatat agacgttgtg gctgttgtag ttgtactcca
     1741 gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt
     1801 tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct
     1861 tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt
     1921 gctgcttcat gtggtcgggg tagcggctga agcactgcac gccgtaggtc agggtggtca
     1981 cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct
     2041 tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt
     2101 cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca
     2161 ccatggcgcg ccgaccggtt tagatcttta ctagtttaag cttcaattgg aagttcctat
     2221 tctttctaga gtataggaac ttcctgcagt aggctagcga agttcctatt ccttttgaag
     2281 tataggaact tcgtcgaggg ggggcccgag cttatcgata atcaacctct ggattacaaa
     2341 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     2401 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     2461 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     2521 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     2581 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     2641 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     2701 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     2761 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     2821 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     2881 cggatctccc tttgggccgc ctccccgcat cgataccgtc gactcgctga tcagcctcga
     2941 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3001 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3061 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3121 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     3181 gaaccagctg gggctcgact agagcatggc tacgtagata agtagcatgg cgggttaatc
     3241 attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     3301 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg
     3361 agcgcgcaga gctttttgca aaagcctagg cctccaaaaa agcctcctca ctacttctgg
     3421 aatagctcag aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca
     3481 tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc
     3541 gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
     3601 cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg catacttctg
     3661 cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca cagctgcatt
     3721 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3781 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3841 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3901 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3961 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4021 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4081 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4141 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4201 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4261 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4321 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4381 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4441 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4501 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4561 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4621 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4681 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4741 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4801 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4861 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4921 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4981 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5041 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5101 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5161 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5221 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5281 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5341 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5401 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5461 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5521 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5581 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5641 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5701 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     5761 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     5821 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     5881 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     5941 tgagagtgca ccattcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     6001 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     6061 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     6121 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     6181 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatctggcta
     6241 gcgatgaccc tgctgattgg ttcgctgacc atttccgggt gcgggacggc gttaccagaa
     6301 actcagaagg ttcgtccaac caaaccgact ctgacggcag tttacgagag agatgatagg
     6361 gtctgcttca gtaagccaga tgctacacaa ttaggcttgt acatattgtc gttagaacgc
     6421 ggctacaatt aatacataac cttatgtatc atacacatac gatttaggtg acactataga
     6481 atacacggaa ttaattctag
//
