LOCUS       sequence-96394-e        6379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cre recombinase from the Cbh promoter and one U6-driven 
            sgRNA control targeting LacZ. AAV backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6379)
  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 6379)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 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(LacZ)-pCBh-Cre-WPRE-hGHpA-ITR
FEATURES             Location/Qualifiers
     source          1..6379
                     /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"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        156..404
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     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        426..501
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        591..876
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        878..1155
                     /label=chicken beta-actin promoter
     intron          1156..1384
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1405..1425
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2452..2478
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    2506..3094
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2559..2579)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2977..2988)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3126..3602
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3325..3344)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3642..3782
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3642..3771
                     /label=AAV2 ITR
     rep_origin      3857..4312
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3944..3963)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4154..4175
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4329..4348)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4448..4470
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4508..4526)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4594..4698
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4699..4767,4768..5559)
                     /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(4917..4936)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5730..6318
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6219..6238
                     /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 caccgtgcga atacgcccac
      421 gcgatgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
      481 aaagtggcac cgagtcggtg cttttttgtt ttagagctag aaatagcaag ttaaaataag
      541 gctagtccgt agcgcgtgcg ccaattctgc agacaaattc tagaggtacc cgttacataa
      601 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      661 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      721 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      781 ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg
      841 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
      901 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
      961 ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg cggggcgggg
     1021 cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc
     1081 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
     1141 agcgcgcggc gggcgggagt cgctgcgacg ctgccttcgc cccgtgcccc gctccgccgc
     1201 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
     1261 ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg
     1321 gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt
     1381 tcaggttgga ccggtgccac catgcccaag aagaagagga aggtgtccaa tctcctgact
     1441 gttcaccaga acctccctgc gctgccagta gatgccacta gcgatgaggt caggaaaaat
     1501 ctcatggata tgtttaggga tagacaggcg ttttctgaac acacctggaa aatgctgctt
     1561 agcgtgtgcc gatcctgggc agcctggtgt aagctgaaca atcgcaaatg gttccccgcc
     1621 gagccggagg acgtgcgcga ttacctgctg tatctccagg caagagggct ggctgtcaag
     1681 actatccagc agcacttggg ccaactgaat atgctgcatc gacgcagcgg gctcccccgg
     1741 cctagcgatt caaacgcagt ctcccttgtt atgaggagaa ttagaaagga aaacgtagat
     1801 gcgggtgaga gggctaagca ggctctcgct tttgagcgga ctgatttcga ccaggtcaga
     1861 tccctgatgg agaacagcga tcggtgccag gacatcagga acctcgcatt tctgggaatt
     1921 gcatataaca cacttctgcg catagctgag atcgcccgga tcagagtgaa agacatcagt
     1981 cgaacggacg gcggccggat gcttattcat attggacgca caaagacatt ggtcagcacc
     2041 gctggcgttg aaaaggcctt gtccctgggc gtaacgaagc tggtggaaag atggatctca
     2101 gtgtccggcg tggctgacga ccctaataat tacttgttct gtcgagtgag aaaaaacgga
     2161 gtcgccgcgc cctctgccac cagccaattg agtacacggg cccttgaagg gatctttgag
     2221 gcaacccacc gactcatata cggagccaag gatgacagtg gccagaggta tctcgcctgg
     2281 tcaggtcatt ctgctagggt gggggccgca cgagacatgg cgcgggcagg agtctccata
     2341 ccagagatta tgcaagctgg aggttggaca aatgtgaaca tcgttatgaa ctatatccgc
     2401 aatcttgact ctgaaaccgg ggccatggtg agactgctcg aagatggtga ctacccatac
     2461 gatgttccag attacgctta agaattcgat atcaagctta tcgataatca acctctggat
     2521 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     2581 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     2641 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     2701 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     2761 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     2821 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     2881 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc
     2941 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3001 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     3061 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga
     3121 tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca
     3181 ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg
     3241 tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga
     3301 agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat
     3361 cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg
     3421 agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga
     3481 gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc
     3541 caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc
     3601 ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga
     3661 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     3721 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     3781 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     3841 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3901 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3961 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4021 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     4081 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4141 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc
     4201 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     4261 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     4321 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     4381 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4441 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     4501 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     4561 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     4621 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     4681 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     4741 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     4801 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     4861 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     4921 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     4981 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5041 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5101 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5161 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5221 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5281 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5341 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5401 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5461 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     5521 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     5581 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     5641 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5701 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5761 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5821 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     5881 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     5941 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6001 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6061 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6121 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6181 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6241 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6301 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6361 tggccttttg ctcacatgt
//
