LOCUS       40924_140        6562 bp DNA     circular SYN 13-MAY-2021
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 6562)
  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 6562)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6562)
  AUTHORS   .
  TITLE     Direct Submission
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     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     promoter        156..396
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        423..498
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        532..979
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             1002..1022
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1020..2048
                     /codon_start=1
                     /label=Cre
                     /note="site-specific recombinase"
                     /translation="VSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             2049..2075
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     CDS             2091..2144
                     /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             2145..2858
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     misc_feature    3150..3738
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     repeat_region   3825..3965
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4040..4495
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4512..4531)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     4631..4653
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4691..4709)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        4777..4881
                     /label=AmpR promoter
     CDS             4882..5739
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5913..6501
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtgaggg cctatttccc atgattcctt
      181 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
      241 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      301 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      361 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggaaga gcgagctctt
      421 ctgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
      481 gtggcaccga gtcggtgctt tttttctaga ctgcagaggg ccctgcgtat gagtgcaagt
      541 gggttttagg accaggatga ggcggggtgg gggtgcctac ctgacgaccg accccgaccc
      601 actggacaag cacccaaccc ccattcccca aattgcgcat cccctatcag agagggggag
      661 gggaaacagg atgcggcgag gcgcgtgcgc actgccagct tcagcaccgc ggacagtgcc
      721 ttcgcccccg cctggcggcg cgcgccaccg ccgcctcagc actgaaggcg cgctgacgtc
      781 actcgccggt cccccgcaaa ctccccttcc cggccacctt ggtcgcgtcc gcgccgccgc
      841 cggcccagcc ggaccgcacc acgcgaggcg cgagataggg gggcacgggc gcgaccatct
      901 gcgctgcggc gccggcgact cagcgctgcc tcagtctgcg gtgggcagcg gaggagtcgt
      961 gtcgtgcctg agagcgcagt cgagaaggta ccgccaccat gcccaagaag aagaggaagg
     1021 tgtccaatct cctgactgtt caccagaacc tccctgcgct gccagtagat gccactagcg
     1081 atgaggtcag gaaaaatctc atggatatgt ttagggatag acaggcgttt tctgaacaca
     1141 cctggaaaat gctgcttagc gtgtgccgat cctgggcagc ctggtgtaag ctgaacaatc
     1201 gcaaatggtt ccccgccgag ccggaggacg tgcgcgatta cctgctgtat ctccaggcaa
     1261 gagggctggc tgtcaagact atccagcagc acttgggcca actgaatatg ctgcatcgac
     1321 gcagcgggct cccccggcct agcgattcaa acgcagtctc ccttgttatg aggagaatta
     1381 gaaaggaaaa cgtagatgcg ggtgagaggg ctaagcaggc tctcgctttt gagcggactg
     1441 atttcgacca ggtcagatcc ctgatggaga acagcgatcg gtgccaggac atcaggaacc
     1501 tcgcatttct gggaattgca tataacacac ttctgcgcat agctgagatc gcccggatca
     1561 gagtgaaaga catcagtcga acggacggcg gccggatgct tattcatatt ggacgcacaa
     1621 agacattggt cagcaccgct ggcgttgaaa aggccttgtc cctgggcgta acgaagctgg
     1681 tggaaagatg gatctcagtg tccggcgtgg ctgacgaccc taataattac ttgttctgtc
     1741 gagtgagaaa aaacggagtc gccgcgccct ctgccaccag ccaattgagt acacgggccc
     1801 ttgaagggat ctttgaggca acccaccgac tcatatacgg agccaaggat gacagtggcc
     1861 agaggtatct cgcctggtca ggtcattctg ctagggtggg ggccgcacga gacatggcgc
     1921 gggcaggagt ctccatacca gagattatgc aagctggagg ttggacaaat gtgaacatcg
     1981 ttatgaacta tatccgcaat cttgactctg aaaccggggc catggtgaga ctgctcgaag
     2041 atggtgacta cccatacgat gttccagatt acgctgctag cggcagtgga gagggcagag
     2101 gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccagtgagc aagggcgagg
     2161 agctgttcac cggggtggta cccatcctgg tcgagctgga cggcgacgta aacggccaca
     2221 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     2281 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     2341 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     2401 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     2461 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     2521 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     2581 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     2641 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     2701 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     2761 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     2821 ccgccgggat cactctcggc atggacgagc tgtacaagtc cggactcaga tctcgagagg
     2881 aggaggagga gacagacagc aggatgcccc acctcgacag ccccggcagc tcccagccga
     2941 gacgctcctt cctctcaagg gtgatcaggg cagcgctacc gttgcagctg cttctgctgc
     3001 tgctgctgct cctggcctgc ctgctacctg cctctgaaga tgactacagc tgcacccagg
     3061 ccaacaactt tgcccgatcc ttctacccca tgctgcggta caccaacggg ccacctccca
     3121 cctaggaatt cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa
     3181 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     3241 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     3301 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     3361 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     3421 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     3481 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     3541 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     3601 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     3661 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     3721 tttgggccgc ctccccgcat cgataccgag cgctaataaa agatctttat tttcattaga
     3781 tctgtgtgtt ggttttttgt gtcacgtgcg gaccgagcgg ccgcaggaac ccctagtgat
     3841 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     3901 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     3961 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     4021 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4081 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     4141 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4201 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     4261 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4321 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     4381 ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     4441 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     4501 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     4561 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4621 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4681 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     4741 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4801 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4861 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     4921 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     4981 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5041 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5101 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5161 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5221 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5281 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5341 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5401 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     5461 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5521 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5581 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     5641 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     5701 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     5761 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5821 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5881 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5941 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6001 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     6061 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6121 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6181 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6241 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6301 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6361 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6421 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6481 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6541 tgctggcctt ttgctcacat gt
//
