LOCUS       sequence-418949-        7191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cre recombinase and Renilla luciferase from the EFS 
            promoter and three U6-driven sgRNAs targeting Kras, p53, and Lkb1. 
            Contains KrasG12D HDR donor. AAV backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7191)
  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 7191)
  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(Kras)-U6-sgRNA(p53)-U6-sgRNA(Lkb1)-pEFS-Rluc-2A-Cre
            -shortPA-KrasG12D_HDRdonor-ITR (AAV-KPL)
FEATURES             Location/Qualifiers
     source          1..7191
                     /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..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        502..750
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        771..846
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        860..1108
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1129..1204
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1259..1470
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1484..2416
                     /codon_start=1
                     /product="luciferase from the anthozoan coelenterate 
                     Renilla reniformis (sea pansy)"
                     /label=Rluc
                     /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     CDS             2432..2488
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2489..2509
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3536..3562
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     repeat_region   4454..4594
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4454..4583
                     /label=AAV2 ITR
     rep_origin      4669..5124
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4756..4775)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4966..4987
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5141..5160)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5260..5282
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5320..5338)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5406..5510
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5511..5579,5580..6371)
                     /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(5729..5748)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6542..7130
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7031..7050
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
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 accgcagcgt tacctctatc gtagttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct tttttggatc cgagggccta tttcccatga ttccttcata tttgcatata
      541 cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta
      601 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      661 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      721 ttatatatct tgtggaaagg acgaaacacc gtgtaatagc tcctgcatgg gttttagagc
      781 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
      841 cggtgctttt tttctagaag agggcctatt tcccatgatt ccttcatatt tgcatatacg
      901 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
      961 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     1021 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     1081 atatatcttg tggaaaggac gaaacaccac tccgagacct tatgccgcgt tttagagcta
     1141 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     1201 gtgctttttt ggtaccaggt cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg
     1261 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc
     1321 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
     1381 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
     1441 tttcgcaacg ggtttgccgc cagaacacag gcgtacggcc accatgactt cgaaagttta
     1501 tgatccagaa caaaggaaac ggatgataac tggtccgcag tggtgggcca gatgtaaaca
     1561 aatgaatgtt cttgattcat ttattaatta ttatgattca gaaaaacatg cagaaaatgc
     1621 tgttattttt ttacatggta acgcggcctc ttcttattta tggcgacatg ttgtgccaca
     1681 tattgagcca gtagcgcggt gtattatacc agaccttatt ggtatgggca aatcaggcaa
     1741 atctggtaat ggttcttata ggttacttga tcattacaaa tatcttactg catggtttga
     1801 acttcttaat ttaccaaaga agatcatttt tgtcggccat gattggggtg cttgtttggc
     1861 atttcattat agctatgagc atcaagataa gatcaaagca atagttcacg ctgaaagtgt
     1921 agtagatgtg attgaatcat gggatgaatg gcctgatatt gaagaagata ttgcgttgat
     1981 caaatctgaa gaaggagaaa aaatggtttt ggagaataac ttcttcgtgg aaaccatgtt
     2041 gccatcaaaa atcatgagaa agttagaacc agaagaattt gcagcatatc ttgaaccatt
     2101 caaagagaaa ggtgaagttc gtcgtccaac attatcatgg cctcgtgaaa tcccgttagt
     2161 aaaaggtggt aaacctgacg ttgtacaaat tgttaggaat tataatgctt atctacgtgc
     2221 aagtgatgat ttaccaaaaa tgtttattga atcggaccca ggattctttt ccaatgctat
     2281 tgttgaaggt gccaagaagt ttcctaatac tgaatttgtc aaagtaaaag gtcttcattt
     2341 ttcgcaagaa gatgcacctg atgaaatggg aaaatatatc aaatcgttcg ttgagcgagt
     2401 tctcaaaaat gaacaagcta gcggaagcgg agccactaac ttctccctgt tgaaacaagc
     2461 aggggatgtc gaagagaatc ccgggccacc caagaagaag aggaaggtgt ccaatctcct
     2521 gactgttcac cagaacctcc ctgcgctgcc agtagatgcc actagcgatg aggtcaggaa
     2581 aaatctcatg gatatgttta gggatagaca ggcgttttct gaacacacct ggaaaatgct
     2641 gcttagcgtg tgccgatcct gggcagcctg gtgtaagctg aacaatcgca aatggttccc
     2701 cgccgagccg gaggacgtgc gcgattacct gctgtatctc caggcaagag ggctggctgt
     2761 caagactatc cagcagcact tgggccaact gaatatgctg catcgacgca gcgggctccc
     2821 ccggcctagc gattcaaacg cagtctccct tgttatgagg agaattagaa aggaaaacgt
     2881 agatgcgggt gagagggcta agcaggctct cgcttttgag cggactgatt tcgaccaggt
     2941 cagatccctg atggagaaca gcgatcggtg ccaggacatc aggaacctcg catttctggg
     3001 aattgcatat aacacacttc tgcgcatagc tgagatcgcc cggatcagag tgaaagacat
     3061 cagtcgaacg gacggcggcc ggatgcttat tcatattgga cgcacaaaga cattggtcag
     3121 caccgctggc gttgaaaagg ccttgtccct gggcgtaacg aagctggtgg aaagatggat
     3181 ctcagtgtcc ggcgtggctg acgaccctaa taattacttg ttctgtcgag tgagaaaaaa
     3241 cggagtcgcc gcgccctctg ccaccagcca attgagtaca cgggcccttg aagggatctt
     3301 tgaggcaacc caccgactca tatacggagc caaggatgac agtggccaga ggtatctcgc
     3361 ctggtcaggt cattctgcta gggtgggggc cgcacgagac atggcgcggg caggagtctc
     3421 cataccagag attatgcaag ctggaggttg gacaaatgtg aacatcgtta tgaactatat
     3481 ccgcaatctt gactctgaaa ccggggccat ggtgagactg ctcgaagatg gtgactaccc
     3541 atacgatgtt ccagattacg cttaaagcgc taataaaaga tctttatttt cattagatct
     3601 gtgtgttggt tttttgtgta agctttggct ccaacacaga tgttcttagg ctacctaact
     3661 tctaactttt aatatccagt caacaaagaa taccgcaagg gtaggtgttg ggatagctgt
     3721 cgacaagctc atgcgggtgt gtccacaggg tatagcgtac tatgcagaat atttgtactg
     3781 agtgaagtca tgatacattc ctttgagagc cattagctgc tacaaaacag taatctggct
     3841 gtttagatca acaagctaaa tgatagaaga tgaaagtact ggtttccatg tatttttatt
     3901 aagtgttgat gagaaagttg taagtgactt acaggttact ctgtacatct gtagtcactg
     3961 aattcggaat atcttagagt tttacacaca aaggtgagtg ttaaaatatt gataaagttt
     4021 ttgataatct tgtgtgagac atgttctaat ttagttgtat tttattattt ttattgtaag
     4081 gcctgctgaa aatgactgag tataaacttg tggtcgtggg cgccgacggc gtgggcaaga
     4141 gcgctttgac gatacagcta attcagaatc actttgtgga tgagtatgat ccaaccatcg
     4201 aggtaacgct gctctacagt ctgcgtgcgc ttgtaaagga cggcagccag ccgctttgaa
     4261 aaagatatca tttttatatt tattagaaaa ttatattgaa agttatttca gttatatgtg
     4321 atgtccttta gttccaaggc tttaaactgg gtgttaggga accataggtg caagaaagtc
     4381 cacttctcat gagagctcac cacagagaaa gaaagtccac ttctcagtaa ccacgtgcgg
     4441 accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4501 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4561 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     4621 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     4681 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4741 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4801 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4861 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4921 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4981 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     5041 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     5101 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     5161 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5221 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5281 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     5341 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     5401 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5461 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5521 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5581 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5641 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5701 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5761 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5821 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5881 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5941 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6001 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6061 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6121 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6181 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6241 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6301 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6361 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6421 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6481 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6541 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6601 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6661 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6721 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6781 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6841 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6901 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6961 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7021 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7081 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7141 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
