LOCUS       40924_145        6362 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses Cre recombinase from the Cbh 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 6362)
  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 6362)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6362)
  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..6362
                     /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..385
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        412..487
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        577..862
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        864..1139
                     /label=chicken beta-actin promoter
     intron          1140..1367
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1388..1408
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1406..2434
                     /codon_start=1
                     /label=Cre
                     /note="site-specific recombinase"
                     /translation="VSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             2435..2461
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     misc_feature    2489..3077
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     polyA_signal    3109..3585
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     repeat_region   3625..3765
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      3840..4295
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4312..4331)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     4431..4453
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4491..4509)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        4577..4681
                     /label=AmpR promoter
     CDS             4682..5539
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5713..6301
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
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 tttgttttag agctagaaat agcaagttaa aataaggcta gtccgtagcg
      541 cgtgcgccaa ttctgcagac aaattctaga ggtacccgtt acataactta cggtaaatgg
      601 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg
      661 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      721 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      781 ctggcattgt gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      841 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
      901 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
      961 gatgggggcg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg
     1021 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
     1081 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
     1141 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
     1201 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
     1261 cgggctgtaa ttagctgagc aagaggtaag ggtttaaggg atggttggtt ggtggggtat
     1321 taatgtttaa ttacctggag cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc
     1381 caccatgccc aagaagaaga ggaaggtgtc caatctcctg actgttcacc agaacctccc
     1441 tgcgctgcca gtagatgcca ctagcgatga ggtcaggaaa aatctcatgg atatgtttag
     1501 ggatagacag gcgttttctg aacacacctg gaaaatgctg cttagcgtgt gccgatcctg
     1561 ggcagcctgg tgtaagctga acaatcgcaa atggttcccc gccgagccgg aggacgtgcg
     1621 cgattacctg ctgtatctcc aggcaagagg gctggctgtc aagactatcc agcagcactt
     1681 gggccaactg aatatgctgc atcgacgcag cgggctcccc cggcctagcg attcaaacgc
     1741 agtctccctt gttatgagga gaattagaaa ggaaaacgta gatgcgggtg agagggctaa
     1801 gcaggctctc gcttttgagc ggactgattt cgaccaggtc agatccctga tggagaacag
     1861 cgatcggtgc caggacatca ggaacctcgc atttctggga attgcatata acacacttct
     1921 gcgcatagct gagatcgccc ggatcagagt gaaagacatc agtcgaacgg acggcggccg
     1981 gatgcttatt catattggac gcacaaagac attggtcagc accgctggcg ttgaaaaggc
     2041 cttgtccctg ggcgtaacga agctggtgga aagatggatc tcagtgtccg gcgtggctga
     2101 cgaccctaat aattacttgt tctgtcgagt gagaaaaaac ggagtcgccg cgccctctgc
     2161 caccagccaa ttgagtacac gggcccttga agggatcttt gaggcaaccc accgactcat
     2221 atacggagcc aaggatgaca gtggccagag gtatctcgcc tggtcaggtc attctgctag
     2281 ggtgggggcc gcacgagaca tggcgcgggc aggagtctcc ataccagaga ttatgcaagc
     2341 tggaggttgg acaaatgtga acatcgttat gaactatatc cgcaatcttg actctgaaac
     2401 cggggccatg gtgagactgc tcgaagatgg tgactaccca tacgatgttc cagattacgc
     2461 ttaagaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag
     2521 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     2581 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     2641 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     2701 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     2761 ttccggaact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     2821 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     2881 gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac
     2941 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     3001 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     3061 ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatctacgg gtggcatccc
     3121 tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc
     3181 cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt
     3241 atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc
     3301 tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc
     3361 tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag
     3421 gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata
     3481 ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa
     3541 ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta
     3601 accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     3661 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     3721 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     3781 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     3841 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3901 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3961 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4021 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     4081 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4141 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     4201 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4261 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     4321 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     4381 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4441 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     4501 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     4561 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4621 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4681 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4741 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4801 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4861 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4921 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4981 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5041 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5101 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5161 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5221 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5281 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5341 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5401 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5461 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5521 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5581 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5641 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5701 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5761 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5821 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     5881 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5941 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6001 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6061 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6121 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6181 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6241 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6301 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6361 gt
//
