LOCUS       sequence-449624-        6016 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV backbone for sgRNA expression. Contains a Lox-flanked handle 
            sequence for Cre-dependent PCR amplification of sgRNAs. Protospacer 
            is cloned between BstXI and BlpI..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6016)
  AUTHORS   Ramani B, Rose IVL, Teyssier N, Pan A, Danner-Bocks S, Sanghal T, 
            Yadanar L, Tian R, Ma K, Palop JJ, Kampmann M
  TITLE     CRISPR screening by AAV episome-sequencing (CrAAVe-seq): a scalable 
            cell-type-specific in vivo platform uncovers neuronal essential 
            genes.
  JOURNAL   Nat Neurosci. 2025 Aug 22. doi: 10.1038/s41593-025-02043-9.
  PUBMED    40847019
REFERENCE   2  (bases 1 to 6016)
  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: "Nat 
            Neurosci. 2025 Aug 22. doi: 10.1038/s41593-025-02043-9."
COMMENT     Sequence Label: pAP215.rAAV.mU6-sgRNA.Handle.EF1a-mTagBFP2
FEATURES             Location/Qualifiers
     source          1..6016
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        795..1107
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     1027..1047
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    complement(1234..1267)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1443..1476
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1490..2668
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1721..2659
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2616..2636
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2694..2720
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             2736..2762
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     regulatory      2778..2787
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2784..3494
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     primer_bind     complement(3561..3581)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3768..3889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3805..3824)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3859..3878
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3929..4069
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3929..4058
                     /label=AAV2 ITR
     rep_origin      4144..4599
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4231..4250)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4441..4462
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4616..4635)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4735..4757
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4795..4813)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4881..4985
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4986..5054,5055..5846)
                     /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(5204..5223)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggccgcac gcgtgatccg acgccgccat ctctaggccc gcgccggccc cctcgcacag
      841 acttgtggga gaagctcggc tactcccctg ccccggttaa tttgcatata atatttccta
      901 gtaactatag aggcttaatg tgcgataaaa gacagataat ctgttctttt taatactagc
      961 tacattttac atgataggct tggatttcta taagagatac aaatactaca ttattatgtt
     1021 aaaaaacagc acaaaaggaa actcacccta actgtaaagt aattgtgtgt tttgagacta
     1081 taaatatccc ttggagacca ccttgttggg atgcatagat gaacggatgt ttaagagcta
     1141 agctggaaac agcatagcaa gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg
     1201 caccgagtcg gtgctttttt ttttttttct agataccgtt cgtatagcat acattatacg
     1261 aagttataca tgtaaacgat cctaggcgtc catatgatgc atggcagcaa ttggccattt
     1321 gtaggcagac gcatcgatca gtacagtcgg atagtcgaac gatcattaca gtcagtcgaa
     1381 tgcaaagtag cgaggcatga cctgtgtact taaagcatga ctggtactga cacgtcgcat
     1441 gctaccgttc gtataatgta tgctatacga agttatggta ccggggcccg gctccggtgc
     1501 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     1561 caattgatcc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     1621 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     1681 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc
     1741 ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg
     1801 gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag
     1861 gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg
     1921 gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc
     1981 tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct
     2041 tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga
     2101 cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc
     2161 gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc
     2221 gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc
     2281 ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc
     2341 gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt
     2401 cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag
     2461 cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca
     2521 cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga
     2581 atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag
     2641 tttttttctt ccatttcagg tgtcgtgagc tagcgctacc ggtcgccacc atgcctgccg
     2701 ctaaacgggt caaactcgac ggcggttctg gaggtcctgc tgcaaagcgc gttaagttgg
     2761 atggagggtc agggggagcc accatggtgt ctaagggcga agagctgatt aaggagaaca
     2821 tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag tgcacatccg
     2881 agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg
     2941 gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc agcaagacct
     3001 tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct gagggcttca
     3061 catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca
     3121 gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcacatcca
     3181 acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgag acgctgtacc
     3241 ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg ggcgggagcc
     3301 atctgatcgc aaacgccaag accacatata gatccaagaa acccgctaag aacctcaaga
     3361 tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc aacaacgaga
     3421 cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct agcaaactgg
     3481 ggcacaagct taattaaaga attcgataat caacctctgg attacaaaat ttgtgaaaga
     3541 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     3601 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     3661 tggttagttc ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     3721 gctcggctgt tgggcactga caattccgtg gtgctcgaga gatctacaac ttgtttattg
     3781 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3841 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttac tgattttgta
     3901 ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc
     3961 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     4021 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     4081 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     4141 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4201 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4261 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4321 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     4381 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4441 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     4501 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     4561 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     4621 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     4681 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4741 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     4801 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     4861 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4921 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4981 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5041 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5101 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5161 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5221 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5281 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5341 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5401 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5461 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5521 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5581 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5641 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     5701 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     5761 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5821 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     5881 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5941 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6001 agatcaaagg atcttc
//
