LOCUS       Exported                6378 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses Cre recombinase from the Cbh promoter and one U6-driven 
            sgRNA targeting the mouse gene NeuN. AAV backbone..
ACCESSION   .
VERSION     .
KEYWORDS    AAV:ITR-U6-sgRNA(NeuN)-pCBh-Cre-WPRE-hGHpA-ITR
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6378)
  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 6378)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6378
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        156..396
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     156..176
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     327..346
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        425..500
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        590..875
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        877..1154
                     /label=chicken beta-actin promoter
     intron          1155..1383
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1404..1424
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2451..2477
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    2505..3093
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2558..2578)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2976..2987)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3125..3601
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3324..3343)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3641..3781
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3641..3770
                     /label=AAV2 ITR
     rep_origin      3856..4311
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3943..3962)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4153..4174
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4328..4347)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4447..4469
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4507..4525)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4593..4697
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4698..5558
                     /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(4916..4935)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5729..6317
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6218..6237
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
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 caccgtcggg gtccctgaac
      421 cggagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
      481 aagtggcacc gagtcggtgc ttttttgttt tagagctaga aatagcaagt taaaataagg
      541 ctagtccgta gcgcgtgcgc caattctgca gacaaattct agaggtaccc gttacataac
      601 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag
      661 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      721 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      781 gtaaatggcc cgcctggcat tgtgcccagt acatgacctt atgggacttt cctacttggc
      841 agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct
      901 tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt attttttaat
      961 tattttgtgc agcgatgggg gcgggggggg ggggggggcg cgcgccaggc ggggcggggc
     1021 ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg
     1081 cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa
     1141 gcgcgcggcg ggcgggagtc gctgcgacgc tgccttcgcc ccgtgccccg ctccgccgcc
     1201 gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg
     1261 gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt taagggatgg
     1321 ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat cacttttttt
     1381 caggttggac cggtgccacc atgcccaaga agaagaggaa ggtgtccaat ctcctgactg
     1441 ttcaccagaa cctccctgcg ctgccagtag atgccactag cgatgaggtc aggaaaaatc
     1501 tcatggatat gtttagggat agacaggcgt tttctgaaca cacctggaaa atgctgctta
     1561 gcgtgtgccg atcctgggca gcctggtgta agctgaacaa tcgcaaatgg ttccccgccg
     1621 agccggagga cgtgcgcgat tacctgctgt atctccaggc aagagggctg gctgtcaaga
     1681 ctatccagca gcacttgggc caactgaata tgctgcatcg acgcagcggg ctcccccggc
     1741 ctagcgattc aaacgcagtc tcccttgtta tgaggagaat tagaaaggaa aacgtagatg
     1801 cgggtgagag ggctaagcag gctctcgctt ttgagcggac tgatttcgac caggtcagat
     1861 ccctgatgga gaacagcgat cggtgccagg acatcaggaa cctcgcattt ctgggaattg
     1921 catataacac acttctgcgc atagctgaga tcgcccggat cagagtgaaa gacatcagtc
     1981 gaacggacgg cggccggatg cttattcata ttggacgcac aaagacattg gtcagcaccg
     2041 ctggcgttga aaaggccttg tccctgggcg taacgaagct ggtggaaaga tggatctcag
     2101 tgtccggcgt ggctgacgac cctaataatt acttgttctg tcgagtgaga aaaaacggag
     2161 tcgccgcgcc ctctgccacc agccaattga gtacacgggc ccttgaaggg atctttgagg
     2221 caacccaccg actcatatac ggagccaagg atgacagtgg ccagaggtat ctcgcctggt
     2281 caggtcattc tgctagggtg ggggccgcac gagacatggc gcgggcagga gtctccatac
     2341 cagagattat gcaagctgga ggttggacaa atgtgaacat cgttatgaac tatatccgca
     2401 atcttgactc tgaaaccggg gccatggtga gactgctcga agatggtgac tacccatacg
     2461 atgttccaga ttacgcttaa gaattcgata tcaagcttat cgataatcaa cctctggatt
     2521 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     2581 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     2641 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     2701 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     2761 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     2821 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     2881 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctat gttgccacct
     2941 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     3001 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     3061 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgagcgctg ctcgagagat
     3121 ctacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac
     3181 tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt
     3241 gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa
     3301 gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc
     3361 ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga
     3421 gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag
     3481 acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc
     3541 accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct
     3601 tctgattttg taggtaacca cgtgcggacc gagcggccgc aggaacccct agtgatggag
     3661 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     3721 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     3781 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     3841 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3901 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3961 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4021 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     4081 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4141 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct
     4201 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4261 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     4321 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     4381 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4441 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     4501 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     4561 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4621 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4681 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4741 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4801 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4861 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4921 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4981 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5041 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5101 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5161 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5221 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5281 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5341 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5401 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5461 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5521 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5581 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5641 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5701 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5761 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5821 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     5881 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5941 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6001 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6061 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6121 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6181 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6241 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6301 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6361 ggccttttgc tcacatgt
//
