LOCUS       sequence-419029-        6361 bp    DNA     circular SYN 15-DEC-2025
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 6361)
  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 6361)
  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(backbone)-pCBh-Cre-WPRE-hGHpA...
FEATURES             Location/Qualifiers
     source          1..6361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   77..206
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        221..469
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     221..241
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     392..411
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        488..563
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        653..938
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        940..1214
                     /label=chicken beta-actin promoter
     intron          1215..1442
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1463..1483
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2510..2536
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    2564..3152
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2617..2637)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3035..3046)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3184..3660
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3383..3402)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3700..3840
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3700..3829
                     /label=AAV2 ITR
     rep_origin      3915..4370
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4002..4021)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4212..4233
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4387..4406)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4506..4528
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4566..4584)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4652..4756
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4757..4825,4826..5617)
                     /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(4975..4994)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5788..6361,1..15)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6277..6296
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
       61 ccttttgctc acatgtcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg
      121 gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt
      181 ggccaactcc atcactaggg gttcctgcgg ccgcacgcgt gagggcctat ttcccatgat
      241 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac
      301 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
      361 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
      421 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gaagagcgag
      481 ctcttctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
      541 aaaaagtggc accgagtcgg tgcttttttg ttttagagct agaaatagca agttaaaata
      601 aggctagtcc gtagcgcgtg cgccaattct gcagacaaat tctagaggta cccgttacat
      661 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      721 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
      781 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
      841 acggtaaatg gcccgcctgg cattgtgccc agtacatgac cttatgggac tttcctactt
      901 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct
      961 gcttcactct ccccatcccc cccccccccc cccccccaat tttgtattta tttatttttt
     1021 aattattttg tgcagcgatg ggggcggggg ggggggggcg cgcgccaggc ggggcggggc
     1081 ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg
     1141 cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa
     1201 gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg
     1261 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg
     1321 acggcccttc tcctccgggc tgtaattagc tgagcaagag gtaagggttt aagggatggt
     1381 tggttggtgg ggtattaatg tttaattacc tggagcacct gcctgaaatc actttttttc
     1441 aggttggacc ggtgccacca tgcccaagaa gaagaggaag gtgtccaatc tcctgactgt
     1501 tcaccagaac ctccctgcgc tgccagtaga tgccactagc gatgaggtca ggaaaaatct
     1561 catggatatg tttagggata gacaggcgtt ttctgaacac acctggaaaa tgctgcttag
     1621 cgtgtgccga tcctgggcag cctggtgtaa gctgaacaat cgcaaatggt tccccgccga
     1681 gccggaggac gtgcgcgatt acctgctgta tctccaggca agagggctgg ctgtcaagac
     1741 tatccagcag cacttgggcc aactgaatat gctgcatcga cgcagcgggc tcccccggcc
     1801 tagcgattca aacgcagtct cccttgttat gaggagaatt agaaaggaaa acgtagatgc
     1861 gggtgagagg gctaagcagg ctctcgcttt tgagcggact gatttcgacc aggtcagatc
     1921 cctgatggag aacagcgatc ggtgccagga catcaggaac ctcgcatttc tgggaattgc
     1981 atataacaca cttctgcgca tagctgagat cgcccggatc agagtgaaag acatcagtcg
     2041 aacggacggc ggccggatgc ttattcatat tggacgcaca aagacattgg tcagcaccgc
     2101 tggcgttgaa aaggccttgt ccctgggcgt aacgaagctg gtggaaagat ggatctcagt
     2161 gtccggcgtg gctgacgacc ctaataatta cttgttctgt cgagtgagaa aaaacggagt
     2221 cgccgcgccc tctgccacca gccaattgag tacacgggcc cttgaaggga tctttgaggc
     2281 aacccaccga ctcatatacg gagccaagga tgacagtggc cagaggtatc tcgcctggtc
     2341 aggtcattct gctagggtgg gggccgcacg agacatggcg cgggcaggag tctccatacc
     2401 agagattatg caagctggag gttggacaaa tgtgaacatc gttatgaact atatccgcaa
     2461 tcttgactct gaaaccgggg ccatggtgag actgctcgaa gatggtgact acccatacga
     2521 tgttccagat tacgcttaag aattcgatat caagcttatc gataatcaac ctctggatta
     2581 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     2641 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     2701 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     2761 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     2821 cacctgtcag ctcctttccg gaactttcgc tttccccctc cctattgcca cggcggaact
     2881 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     2941 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg
     3001 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     3061 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     3121 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc
     3181 tacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact
     3241 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg
     3301 tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag
     3361 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct
     3421 tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag
     3481 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga
     3541 cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca
     3601 ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt
     3661 ctgattttgt aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt
     3721 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     3781 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     3841 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     3901 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3961 gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
     4021 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4081 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     4141 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4201 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta
     4261 ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
     4321 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     4381 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     4441 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4501 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     4561 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     4621 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4681 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4741 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4801 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     4861 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4921 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4981 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5041 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5101 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5161 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5221 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     5281 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     5341 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     5401 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     5461 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     5521 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5581 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5641 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5701 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5761 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5821 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5881 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5941 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6001 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6061 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6121 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6181 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6241 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6301 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6361 g
//
