LOCUS       sequence-417789-        7416 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV genome backbone with a CROP-seq-like design for indirect capture
            of gRNA sequences.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7416)
  AUTHORS   Santinha AJ, Klingler E, Kuhn M, Farouni R, Lagler S, Kalamakis G, 
            Lischetti U, Jabaudon D, Platt RJ
  TITLE     Transcriptional linkage analysis with in vivo AAV-Perturb-seq.
  JOURNAL   Nature. 2023 Oct;622(7982):367-375. doi: 10.1038/s41586-023-06570-y.
            Epub 2023 Sep 20.
  PUBMED    37730998
REFERENCE   2  (bases 1 to 7416)
  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"; doi: 
            "10.1038/s41586-023-06570-y"; journalName: "Nature"; date: 
            "2023-10"; volume: "622"; issue: "7982"; pages: "367-375"
COMMENT     Sequence Label: pAS006
FEATURES             Location/Qualifiers
     source          1..7416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          227..454
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             475..495
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1522..1548
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1564..1617
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1618..1731
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             join(1783..1785,1786..2496)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1783..1785,1786..2496)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1825..1846)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2086..2105)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2433..2454
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2847..2867)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3265..3276)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        3386..3634
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3386..3406
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3557..3576
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     3971..3993
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3985..4002
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3986..4002
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        4025..4100
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   4204..4344
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4204..4333
                     /label=AAV2 ITR
     rep_origin      4419..4874
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4506..4525)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4716..4737
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5010..5032
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5070..5088)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5156..5260
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5261..5329,5330..6121)
                     /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(5479..5498)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6292..6880
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6781..6800
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6942..7071
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        7121..7406
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt
       61 tatttatttt ttaattattt tgtgcagcga tgggggcgag gggcggggcg gggcgaggcg
      121 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
      181 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
      241 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
      301 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
      361 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta
      421 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catgcccaag
      481 aagaagagga aggtgtccaa tctcctgact gttcaccaga acctccctgc gctgccagta
      541 gatgccacta gcgatgaggt caggaaaaat ctcatggata tgtttaggga tagacaggcg
      601 ttttctgaac acacctggaa aatgctgctt agcgtgtgcc gatcctgggc agcctggtgt
      661 aagctgaaca atcgcaaatg gttccccgcc gagccggagg acgtgcgcga ttacctgctg
      721 tatctccagg caagagggct ggctgtcaag actatccagc agcacttggg ccaactgaat
      781 atgctgcatc gacgcagcgg gctcccccgg cctagcgatt caaacgcagt ctcccttgtt
      841 atgaggagaa ttagaaagga aaacgtagat gcgggtgaga gggctaagca ggctctcgct
      901 tttgagcgga ctgatttcga ccaggtcaga tccctgatgg agaacagcga tcggtgccag
      961 gacatcagga acctcgcatt tctgggaatt gcatataaca cacttctgcg catagctgag
     1021 atcgcccgga tcagagtgaa agacatcagt cgaacggacg gcggccggat gcttattcat
     1081 attggacgca caaagacatt ggtcagcacc gctggcgttg aaaaggcctt gtccctgggc
     1141 gtaacgaagc tggtggaaag atggatctca gtgtccggcg tggctgacga ccctaataat
     1201 tacttgttct gtcgagtgag aaaaaacgga gtcgccgcgc cctctgccac cagccaattg
     1261 agtacacggg cccttgaagg gatctttgag gcaacccacc gactcatata cggagccaag
     1321 gatgacagtg gccagaggta tctcgcctgg tcaggtcatt ctgctagggt gggggccgca
     1381 cgagacatgg cgcgggcagg agtctccata ccagagatta tgcaagctgg aggttggaca
     1441 aatgtgaaca tcgttatgaa ctatatccgc aatcttgact ctgaaaccgg ggccatggtg
     1501 agactgctcg aagatggtga ctacccatac gatgttccag attacgctgc tagcggcagt
     1561 ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg
     1621 gacgaaaaga ccacaggatg gcgaggagga cacgtcgtcg agggactggc aggagagctg
     1681 gaacagctgc gggctagact ggaacaccat cctcagggac agcgagagcc aggaggaggt
     1741 ggaagcggag gaggaggaag cggaggagga ggtagcggat ccgtgagcaa gggcgaggag
     1801 ctgttcaccg gggtggtacc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1861 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1921 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1981 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2041 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2101 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2161 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2221 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2281 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2341 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     2401 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2461 gccgggatca ctctcggcat ggacgagctg tacaagtccg gactcagatc tcgagaggag
     2521 gaggaggaga cagacagcag gatgccccac ctcgacagcc ccggcagctc ccagcctaga
     2581 cgctccttcc tctcaagggt gatcagggca gcgctaccgt tgcagctgct tctgctgctg
     2641 ctgctgctcc tggcctgcct gctacctgcc tctgaagatg actacagctg cacccaggcc
     2701 aacaactttg cccgatcctt ctaccccatg ctgcggtaca ccaacgggcc acctcccacc
     2761 taagcctagg aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt
     2821 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     2881 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     2941 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     3001 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     3061 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     3121 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     3181 tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg gattctgcgc
     3241 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     3301 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     3361 tccctttggg ccgcctccca cgcgtgaggg cctatttccc atgattcctt catatttgca
     3421 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     3481 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     3541 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     3601 ggctttatat atcttgtgga aaggacgaaa caccggagac gcgcttgtct gtaagcggat
     3661 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     3721 cttaactatg cggcatcaga gcagattgta ctgagagtgc accattaact ataacggtcc
     3781 taaggtagcg aaatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     3841 aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     3901 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     3961 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattcga gctcgggcgt
     4021 ctctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     4081 aagtggcacc gagtcggtgc tttttttcta gaatgcaatc gataccgagc gctaataaaa
     4141 gatctttatt ttcattagat ctgtgtgttg gttttttgtg tcacgtgcgg accgagcggc
     4201 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     4261 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     4321 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     4381 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     4441 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     4501 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4561 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4621 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4681 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4741 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     4801 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     4861 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4921 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4981 atccgcttac agacaagctg tgaccttctc cgggagctgc atgtgtcaga ggttttcacc
     5041 gtcatcaccg aaacgcgcga tacgaaaggg cctcgtgata cgcctatttt tataggttaa
     5101 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5161 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5221 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5281 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5341 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5401 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5461 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5521 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5581 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5641 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5701 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5761 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5821 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     5881 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5941 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6001 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6061 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6121 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6181 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6241 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6301 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6361 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6421 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6481 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6541 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6601 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6661 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6721 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6781 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6841 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6901 tttacggttc ctggcctttt gctggccttt tgctcacatg tcctgcaggc agctgcgcgc
     6961 tcgctcgctc actgaggccg cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga
     7021 gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc tgcggccgca
     7081 cgcgggaggg cctattctgc ggacaaatgc tagaggtacc cgttacataa cttacggtaa
     7141 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata gtaacgccaa
     7201 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     7261 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     7321 ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     7381 acgtattagt catcgctatt accatggtcg aggtga
//
