LOCUS       sequence-490567-        6553 bp    DNA     circular SYN 24-OCT-2025
DEFINITION  Expresses Cre recombinase and KASH-tagged EGFP from the hSyn 
            promoter and one U6-driven sgRNA. AAV backbone with SapI spacer for 
            sgRNA cloning..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6553)
  AUTHORS   Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, 
            Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, 
            Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A,
            Feng G, Sharp PA, Zhang F
  TITLE     CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.
  JOURNAL   Cell. 2014 Sep 24. pii: S0092-8674(14)01163-5. doi: 
            10.1016/j.cell.2014.09.014.
  PUBMED    25263330
REFERENCE   2  (bases 1 to 6553)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 24, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2014 
            Sep 24. pii: S0092-8674(14)01163-5. doi: 
            10.1016/j.cell.2014.09.014."
COMMENT     Sequence Label: 
            AAV:ITR-U6-sgRNA(NeuN)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR
FEATURES             Location/Qualifiers
     source          1..6553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        523..970
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             993..1013
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2040..2066
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2082..2135
                     /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             join(2136..2138,2139..2849)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2136..2138,2139..2849)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2178..2199)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2439..2458)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2786..2807
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3141..3729
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3194..3214)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3612..3623)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3816..3956
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3816..3945
                     /label=AAV2 ITR
     rep_origin      4031..4486
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4118..4137)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4328..4349
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4503..4522)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4622..4644
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4682..4700)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4768..4872
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4873..4941,4942..5733)
                     /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(5091..5110)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5904..6492
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6393..6412
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accgtcgggg tccctgaacc ggagttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct ttttttctag actgcagagg gccctgcgta tgagtgcaag tgggttttag
      541 gaccaggatg aggcggggtg ggggtgccta cctgacgacc gaccccgacc cactggacaa
      601 gcacccaacc cccattcccc aaattgcgca tcccctatca gagaggggga ggggaaacag
      661 gatgcggcga ggcgcgtgcg cactgccagc ttcagcaccg cggacagtgc cttcgccccc
      721 gcctggcggc gcgcgccacc gccgcctcag cactgaaggc gcgctgacgt cactcgccgg
      781 tcccccgcaa actccccttc ccggccacct tggtcgcgtc cgcgccgccg ccggcccagc
      841 cggaccgcac cacgcgaggc gcgagatagg ggggcacggg cgcgaccatc tgcgctgcgg
      901 cgccggcgac tcagcgctgc ctcagtctgc ggtgggcagc ggaggagtcg tgtcgtgcct
      961 gagagcgcag tcgagaaggt accgccacca tgcccaagaa gaagaggaag gtgtccaatc
     1021 tcctgactgt tcaccagaac ctccctgcgc tgccagtaga tgccactagc gatgaggtca
     1081 ggaaaaatct catggatatg tttagggata gacaggcgtt ttctgaacac acctggaaaa
     1141 tgctgcttag cgtgtgccga tcctgggcag cctggtgtaa gctgaacaat cgcaaatggt
     1201 tccccgccga gccggaggac gtgcgcgatt acctgctgta tctccaggca agagggctgg
     1261 ctgtcaagac tatccagcag cacttgggcc aactgaatat gctgcatcga cgcagcgggc
     1321 tcccccggcc tagcgattca aacgcagtct cccttgttat gaggagaatt agaaaggaaa
     1381 acgtagatgc gggtgagagg gctaagcagg ctctcgcttt tgagcggact gatttcgacc
     1441 aggtcagatc cctgatggag aacagcgatc ggtgccagga catcaggaac ctcgcatttc
     1501 tgggaattgc atataacaca cttctgcgca tagctgagat cgcccggatc agagtgaaag
     1561 acatcagtcg aacggacggc ggccggatgc ttattcatat tggacgcaca aagacattgg
     1621 tcagcaccgc tggcgttgaa aaggccttgt ccctgggcgt aacgaagctg gtggaaagat
     1681 ggatctcagt gtccggcgtg gctgacgacc ctaataatta cttgttctgt cgagtgagaa
     1741 aaaacggagt cgccgcgccc tctgccacca gccaattgag tacacgggcc cttgaaggga
     1801 tctttgaggc aacccaccga ctcatatacg gagccaagga tgacagtggc cagaggtatc
     1861 tcgcctggtc aggtcattct gctagggtgg gggccgcacg agacatggcg cgggcaggag
     1921 tctccatacc agagattatg caagctggag gttggacaaa tgtgaacatc gttatgaact
     1981 atatccgcaa tcttgactct gaaaccgggg ccatggtgag actgctcgaa gatggtgact
     2041 acccatacga tgttccagat tacgctgcta gcggcagtgg agagggcaga ggaagtctgc
     2101 taacatgcgg tgacgtcgag gagaatcctg gcccagtgag caagggcgag gagctgttca
     2161 ccggggtggt acccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     2221 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     2281 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     2341 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     2401 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     2461 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     2521 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     2581 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     2641 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2701 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     2761 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2821 tcactctcgg catggacgag ctgtacaagt ccggactcag atctcgagag gaggaggagg
     2881 agacagacag caggatgccc cacctcgaca gccccggcag ctcccagccg agacgctcct
     2941 tcctctcaag ggtgatcagg gcagcgctac cgttgcagct gcttctgctg ctgctgctgc
     3001 tcctggcctg cctgctacct gcctctgaag atgactacag ctgcacccag gccaacaact
     3061 ttgcccgatc cttctacccc atgctgcggt acaccaacgg gccacctccc acctaggaat
     3121 tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     3181 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     3241 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     3301 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     3361 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     3421 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     3481 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     3541 cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct
     3601 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     3661 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     3721 cctccccgca tcgataccga gcgctaataa aagatcttta ttttcattag atctgtgtgt
     3781 tggttttttg tgtcacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc
     3841 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     3901 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     3961 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     4021 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4081 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     4141 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4201 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     4261 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4321 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     4381 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     4441 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     4501 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     4561 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4621 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     4681 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     4741 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4801 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4861 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4921 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4981 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5041 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5101 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5161 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5221 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5281 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5341 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5401 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5461 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5521 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5581 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5641 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5701 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5761 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5821 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5881 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5941 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6001 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6061 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6121 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6181 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6241 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6301 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6361 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6421 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6481 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6541 tttgctcaca tgt
//
