LOCUS       sequence-417804-        6551 bp    DNA     circular SYN 14-APR-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 6551)
  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 6551)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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(backbone)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR
FEATURES             Location/Qualifiers
     source          1..6551
                     /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        412..487
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        521..968
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             991..1011
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2038..2064
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2080..2133
                     /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(2134..2136,2137..2847)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2134..2136,2137..2847)
                     /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(2176..2197)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2437..2456)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2784..2805
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3139..3727
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3192..3212)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3610..3621)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3814..3954
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3814..3943
                     /label=AAV2 ITR
     rep_origin      4029..4484
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4116..4135)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4326..4347
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4501..4520)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4620..4642
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4680..4698)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4766..4870
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4871..4939,4940..5731)
                     /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(5089..5108)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5902..6490
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6391..6410
                     /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 accggaagag cgagctcttc tgttttagag
      421 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      481 tcggtgcttt ttttctagac tgcagagggc cctgcgtatg agtgcaagtg ggttttagga
      541 ccaggatgag gcggggtggg ggtgcctacc tgacgaccga ccccgaccca ctggacaagc
      601 acccaacccc cattccccaa attgcgcatc ccctatcaga gagggggagg ggaaacagga
      661 tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc
      721 ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc
      781 ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg
      841 gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg cgctgcggcg
      901 ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga
      961 gagcgcagtc gagaaggtac cgccaccatg cccaagaaga agaggaaggt gtccaatctc
     1021 ctgactgttc accagaacct ccctgcgctg ccagtagatg ccactagcga tgaggtcagg
     1081 aaaaatctca tggatatgtt tagggataga caggcgtttt ctgaacacac ctggaaaatg
     1141 ctgcttagcg tgtgccgatc ctgggcagcc tggtgtaagc tgaacaatcg caaatggttc
     1201 cccgccgagc cggaggacgt gcgcgattac ctgctgtatc tccaggcaag agggctggct
     1261 gtcaagacta tccagcagca cttgggccaa ctgaatatgc tgcatcgacg cagcgggctc
     1321 ccccggccta gcgattcaaa cgcagtctcc cttgttatga ggagaattag aaaggaaaac
     1381 gtagatgcgg gtgagagggc taagcaggct ctcgcttttg agcggactga tttcgaccag
     1441 gtcagatccc tgatggagaa cagcgatcgg tgccaggaca tcaggaacct cgcatttctg
     1501 ggaattgcat ataacacact tctgcgcata gctgagatcg cccggatcag agtgaaagac
     1561 atcagtcgaa cggacggcgg ccggatgctt attcatattg gacgcacaaa gacattggtc
     1621 agcaccgctg gcgttgaaaa ggccttgtcc ctgggcgtaa cgaagctggt ggaaagatgg
     1681 atctcagtgt ccggcgtggc tgacgaccct aataattact tgttctgtcg agtgagaaaa
     1741 aacggagtcg ccgcgccctc tgccaccagc caattgagta cacgggccct tgaagggatc
     1801 tttgaggcaa cccaccgact catatacgga gccaaggatg acagtggcca gaggtatctc
     1861 gcctggtcag gtcattctgc tagggtgggg gccgcacgag acatggcgcg ggcaggagtc
     1921 tccataccag agattatgca agctggaggt tggacaaatg tgaacatcgt tatgaactat
     1981 atccgcaatc ttgactctga aaccggggcc atggtgagac tgctcgaaga tggtgactac
     2041 ccatacgatg ttccagatta cgctgctagc ggcagtggag agggcagagg aagtctgcta
     2101 acatgcggtg acgtcgagga gaatcctggc ccagtgagca agggcgagga gctgttcacc
     2161 ggggtggtac ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2221 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     2281 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     2341 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     2401 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     2461 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2521 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     2581 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     2641 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     2701 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     2761 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     2821 actctcggca tggacgagct gtacaagtcc ggactcagat ctcgagagga ggaggaggag
     2881 acagacagca ggatgcccca cctcgacagc cccggcagct cccagccgag acgctccttc
     2941 ctctcaaggg tgatcagggc agcgctaccg ttgcagctgc ttctgctgct gctgctgctc
     3001 ctggcctgcc tgctacctgc ctctgaagat gactacagct gcacccaggc caacaacttt
     3061 gcccgatcct tctaccccat gctgcggtac accaacgggc cacctcccac ctaggaattc
     3121 gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     3181 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     3241 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     3301 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     3361 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3421 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3481 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     3541 tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac gtccttctgc
     3601 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3661 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     3721 tccccgcatc gataccgagc gctaataaaa gatctttatt ttcattagat ctgtgtgttg
     3781 gttttttgtg tcacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca
     3841 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     3901 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     3961 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     4021 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4081 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     4141 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4201 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     4261 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4321 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     4381 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     4441 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     4501 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     4561 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4621 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     4681 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     4741 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4801 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4861 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4921 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4981 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5041 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5101 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5161 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5221 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5281 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5341 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5401 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     5461 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     5521 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     5581 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     5641 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5701 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     5761 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5821 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5881 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5941 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6001 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6061 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6121 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6181 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6241 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6301 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6361 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6421 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6481 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6541 tgctcacatg t
//
