LOCUS       Exported                6363 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 6363)
  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 6363)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6363
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        228..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     misc_RNA        complement(603..678)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(707..947)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     complement(757..776)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(927..947)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   962..1091
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(1153..1741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1233..1252)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1912..2772)
                     /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     2535..2554
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2773..2877)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2945..2963
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3001..3023)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3123..3142
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3159..3614)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3296..3317)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3508..3527
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3689..3829
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3700..3829
                     /label=AAV2 ITR
     polyA_signal    3869..4345
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     4127..4146
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    4377..4965
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             4483..4494
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     4892..4912
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4993..5019)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(6046..6066)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          6087..6314
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     promoter        complement(6315..226)
                     /label=chicken beta-actin promoter
ORIGIN
        1 gccataaaag gaaactttcg gagcgcgccg ctctgattgg ctgccgccgc acctctccgc
       61 ctcgccccgc cccgcccctc gccccgcccc gccccgcctg gcgcgcgccc cccccccccc
      121 gcccccatcg ctgcacaaaa taattaaaaa ataaataaat acaaaattgg gggtggggag
      181 gggggggaga tggggagagt gaagcagaac gtggggctca cctcgaccat ggtaatagcg
      241 atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat gtactgggca
      301 caatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact tggcatatga
      361 tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca ttgacgtcaa
      421 tggaaagtcc ctattggcgt tactattgac gtcaatgggc gggggtcgtt gggcggtcag
      481 ccaggcgggc catttaccgt aagttatgta acgggtacct ctagaatttg tctgcagaat
      541 tggcgcacgc gctacggact agccttattt taacttgcta tttctagctc taaaacaaaa
      601 aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc
      661 tatttctagc tctaaaactc cggttcaggg accccgacgg tgtttcgtcc tttccacaag
      721 atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg atagtccatt
      781 ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct acgtcacgta
      841 ttttgtacta atatctttgt gtttacagtc aaattaattc caattatctc tctaacagcc
      901 ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctcacg cgtgcggccg
      961 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     1021 gccgggcgac caaaggtcgc ccgacgcccg ggcggcctca gtgagcgagc gagcgcgcag
     1081 ctgcctgcag gacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     1141 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     1201 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     1261 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     1321 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     1381 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     1441 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     1501 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     1561 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     1621 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     1681 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     1741 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     1801 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     1861 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     1921 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     1981 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     2041 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     2101 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     2161 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     2221 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     2281 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     2341 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     2401 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     2461 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     2521 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     2581 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     2641 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     2701 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     2761 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     2821 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     2881 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     2941 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     3001 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     3061 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     3121 tgcggcatca gagcagattg tactgagagt gcaccataaa attgtaaacg ttaatatttt
     3181 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat agaccgaaat
     3241 cggcaaaatc ccttataaat caaaagaata gcccgagata gagttgagtg ttgttccagt
     3301 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     3361 ctatcagggc gatggcccac tacgtgaacc atcacccaaa tcaagttttt tggggtcgag
     3421 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     3481 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctaaggc
     3541 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     3601 gctacagggc gcgtactatg gttgctttga cgtatgcggt gtgaaatacc gcacagatgc
     3661 gtaaggagaa aataccgcat caggcgcccc tgcaggcagc tgcgcgctcg ctcgctcact
     3721 gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc
     3781 gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgctcg
     3841 gtccgcacgt ggttacctac aaaatcagaa ggacagggaa gggagcagtg gttcacgcct
     3901 gtaatcccag caatttggga ggccaaggtg ggtagatcac ctgagattag gagttggaga
     3961 ccagcctggc caatatggtg aaaccccgtc tctaccaaaa aaacaaaaat tagctgagcc
     4021 tggtcatgca tgcctggaat cccaacaact cgggaggctg aggcaggaga atcgcttgaa
     4081 cccaggaggc ggagattgca gtgagccaag attgtgccac tgcactccag cttggttccc
     4141 aatagacccc gcaggcccta caggttgtct tcccaacttg ccccttgctc cataccaccc
     4201 ccctccaccc cataatatta tagaaggaca cctagtcaga caaaatgatg caacttaatt
     4261 ttattaggac aaggctggtg ggcactggag tggcaacttc cagggccagg agaggcactg
     4321 gggaggggtc acagggatgc cacccgtaga tctctcgagc agcgctcggt atcgatgcgg
     4381 ggaggcggcc caaagggaga tccgactcgt ctgagggcga aggcgaagac gcggaagagg
     4441 ccgcagagcc ggcagcaggc cgcgggaagg aaggtccgct ggattgaggg ccgaagggac
     4501 gtagcagaag gacgtcccgc gcagaatcca ggtggcaaca taggcgagca gccaaggaaa
     4561 ggacgatgat ttccccgaca acaccacgga attgtcagtg cccaacagcc gagcccctgt
     4621 ccagcagcgg gcaaggcagg cggcgatgag ttccgccgtg gcaataggga gggggaaagc
     4681 gaaagttccg gaaaggagct gacaggtggt ggcaatgccc caaccagtgg gggttgcgtc
     4741 agcaaacaca gtgcacacca cgccacgttg cctgacaacg ggccacaact cctcataaag
     4801 agacagcaac caggatttat acaaggagga gaaaatgaaa gccatacggg aagcaatagc
     4861 atgatacaaa ggcattaaag cagcgtatcc acatagcgta aaaggagcaa catagttaag
     4921 aataccagtc aatctttcac aaattttgta atccagaggt tgattatcga taagcttgat
     4981 atcgaattct taagcgtaat ctggaacatc gtatgggtag tcaccatctt cgagcagtct
     5041 caccatggcc ccggtttcag agtcaagatt gcggatatag ttcataacga tgttcacatt
     5101 tgtccaacct ccagcttgca taatctctgg tatggagact cctgcccgcg ccatgtctcg
     5161 tgcggccccc accctagcag aatgacctga ccaggcgaga tacctctggc cactgtcatc
     5221 cttggctccg tatatgagtc ggtgggttgc ctcaaagatc ccttcaaggg cccgtgtact
     5281 caattggctg gtggcagagg gcgcggcgac tccgtttttt ctcactcgac agaacaagta
     5341 attattaggg tcgtcagcca cgccggacac tgagatccat ctttccacca gcttcgttac
     5401 gcccagggac aaggcctttt caacgccagc ggtgctgacc aatgtctttg tgcgtccaat
     5461 atgaataagc atccggccgc cgtccgttcg actgatgtct ttcactctga tccgggcgat
     5521 ctcagctatg cgcagaagtg tgttatatgc aattcccaga aatgcgaggt tcctgatgtc
     5581 ctggcaccga tcgctgttct ccatcaggga tctgacctgg tcgaaatcag tccgctcaaa
     5641 agcgagagcc tgcttagccc tctcacccgc atctacgttt tcctttctaa ttctcctcat
     5701 aacaagggag actgcgtttg aatcgctagg ccgggggagc ccgctgcgtc gatgcagcat
     5761 attcagttgg cccaagtgct gctggatagt cttgacagcc agccctcttg cctggagata
     5821 cagcaggtaa tcgcgcacgt cctccggctc ggcggggaac catttgcgat tgttcagctt
     5881 acaccaggct gcccaggatc ggcacacgct aagcagcatt ttccaggtgt gttcagaaaa
     5941 cgcctgtcta tccctaaaca tatccatgag atttttcctg acctcatcgc tagtggcatc
     6001 tactggcagc gcagggaggt tctggtgaac agtcaggaga ttggacacct tcctcttctt
     6061 cttgggcatg gtggcaccgg tccaacctga aaaaaagtga tttcaggcag gtgctccagg
     6121 taattaaaca ttaatacccc accaaccaac catcccttaa acccttacct cttgctcagc
     6181 taattacagc ccggaggaga agggccgtcc cgcccgctca cctgtgggag taacgcggtc
     6241 agtcagagcc ggggcgggcg gcgcgaggcg gcggcggagc ggggcacggg gcgaaggcag
     6301 cgcgcagcga ctcccgcccg ccgcgcgctt cgctttttat agggccgccg ccgccgccgc
     6361 ctc
//
