LOCUS       sequence-308177-        6573 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for Cre-dependent expression of EGFP tagged KASH protein 
            domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6573)
  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 6573)
  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-FLEX-EGFP-KASH
FEATURES             Location/Qualifiers
     source          1..6573
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     primer_bind     962..984
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1046..1065
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(1116..1149)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1200..1233)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1509..2219,2220..2222,2223..2225))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1509..2219,2220..2222,2223..2225))
                     /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(1551..1572)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1900..1919
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2159..2180
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2251..2284
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    2335..2368
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2393..2981
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2446..2466)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2864..2875)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3008..3025)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3014..3238
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(3448..3777)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3462..3597
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3762..3782
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(4023..4611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4103..4122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4782..5573,5574..5642))
                     /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     5405..5424
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5643..5747)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5815..5833
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5871..5893)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5993..6012
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6226..6245
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6535..6553
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6535..6552
                     /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 gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
      601 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
      661 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
      721 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
      781 gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg
      841 tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg
      901 ggagcggctc ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg
      961 gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc
     1021 ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt
     1081 ggcaaagaat tggatcctct agagtcgact ccggaataac ttcgtatagg atactttata
     1141 cgaagttatg cagaatggta gctggattgt agctgctatt agcaatatga aacctcttaa
     1201 taacttcgta tagcatacat tatacgaagt tatggcgcgc cctaggtggg aggtggcccg
     1261 ttggtgtacc gcagcatggg gtagaaggat cgggcaaagt tgttggcctg ggtgcagctg
     1321 tagtcatctt cagaggcagg tagcaggcag gccaggagca gcagcagcag cagaagcagc
     1381 tgcaacggta gcgctgccct gatcaccctt gagaggaagg agcgtctcgg ctgggagctg
     1441 ccggggctgt cgaggtgggg catcctgctg tctgtctcct cctcctcctc tcgagatctg
     1501 agtccggact tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc
     1561 agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc
     1621 aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag
     1681 tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg
     1741 atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc
     1801 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg
     1861 ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc
     1921 ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg
     1981 tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc
     2041 acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc
     2101 ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg
     2161 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc
     2221 accatgcggc cgctgcagac cggtgctagc ataacttcgt ataaagtatc ctatacgaag
     2281 ttatttgcct taacccagaa attatcactg ttattcttta gaatggtgca aagaataact
     2341 tcgtataatg tatgctatac gaagttatga attcgatatc aagcttatcg ataatcaacc
     2401 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     2461 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     2521 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     2581 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     2641 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     2701 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     2761 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     2821 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     2881 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     2941 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgactcgc
     3001 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3061 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3121 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3181 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     3241 tctgaggcgg aaagaaccag ctggggctcg actagagcat ggctacgtag ataagtagca
     3301 tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct
     3361 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct
     3421 cagtgagcga gcgagcgcgc agagcttttt gcaaaagcct aggcctccaa aaaagcctcc
     3481 tcactacttc tggaatagct cagaggccga ggcggcctcg gcctctgcat aaataaaaaa
     3541 aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg
     3601 cggagttagg ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct
     3661 gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga gatgcatgct
     3721 ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt
     3781 ccacagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3841 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3901 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3961 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4021 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4081 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4141 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4201 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4261 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4321 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4381 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4441 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4501 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4561 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4621 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4681 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4741 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4801 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4861 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4921 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4981 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5041 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5101 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5161 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5221 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5281 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5341 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5401 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5461 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5521 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5581 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5641 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5701 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5761 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     5821 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     5881 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     5941 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     6001 gagcagattg tactgagagt gcaccattcg acgctctccc ttatgcgact cctgcattag
     6061 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     6121 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     6181 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     6241 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     6301 gaggatctgg ctagcgatga ccctgctgat tggttcgctg accatttccg ggtgcgggac
     6361 ggcgttacca gaaactcaga aggttcgtcc aaccaaaccg actctgacgg cagtttacga
     6421 gagagatgat agggtctgct tcagtaagcc agatgctaca caattaggct tgtacatatt
     6481 gtcgttagaa cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag
     6541 gtgacactat agaatacacg gaattaattc tag
//
