LOCUS       sequence-417794-        6004 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV genome to express mTagBF2 under control of EF1a. Use for 
            tri-color experiments to define injection conditions for 
            AAV-Perturb-seq.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6004)
  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 6004)
  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: pAS133
FEATURES             Location/Qualifiers
     source          1..6004
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     403..423
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      470..479
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             476..1186
                     /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(1537..1557)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1955..1966)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2116..2250
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2153..2172)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2207..2226
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   2274..2414
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2274..2403
                     /label=AAV2 ITR
     rep_origin      2489..2944
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2576..2595)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2786..2807
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2961..2980)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3080..3102
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3140..3158)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3226..3330
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3331..3399,3400..4191)
                     /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(3549..3568)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4362..4950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4851..4870
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5012..5141
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        join(5281..6004,1..455)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          join(5512..6004,1..446)
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
ORIGIN
        1 tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg
       61 cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc
      121 ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct
      181 cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt
      241 tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt
      301 ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct
      361 ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg
      421 ttcaaagttt ttttcttcca tttcaggtgt cgtgaggcgc gccaccggtg ccaccatggt
      481 gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
      541 cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
      601 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
      661 ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga
      721 cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga
      781 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
      841 cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact
      901 cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg aaggcagaaa
      961 cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacgcca agaccacata
     1021 tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag
     1081 actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt
     1141 ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattccg gactcagatc
     1201 tcgagaggag gaggaggaga cagacagcag gatgccccac ctcgacagcc ccggcagctc
     1261 ccagcctaga cgctccttcc tctcaagggt gatcagggca gcgctaccgt tgcagctgct
     1321 tctgctgctg ctgctgctcc tggcctgcct gctacctgcc tctgaagatg actacagctg
     1381 cacccaggcc aacaactttg cccgatcctt ctaccccatg ctgcggtaca ccaacgggcc
     1441 acctcccacc taagcctagg aattcgatat caagcttatc gataatcaac ctctggatta
     1501 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     1561 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     1621 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     1681 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     1741 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     1801 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     1861 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg
     1921 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     1981 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     2041 gagtcggatc tccctttggg ccgcctccca cgcgggaggg cctattctgc agacaaatgc
     2101 tagaatgcaa tcgataactt gtttattgca gcttataatg gttacaaata aagcaatagc
     2161 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     2221 ctcatcaatg tatcttatca tgtctggatc tcacgtgcgg accgagcggc cgcaggaacc
     2281 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     2341 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     2401 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2461 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     2521 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     2581 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2641 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2701 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2761 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2821 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     2881 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     2941 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     3001 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3061 agacaagctg tgaccttctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3121 aaacgcgcga tacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     3181 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3241 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3301 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3361 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3421 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3481 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3541 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3601 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3661 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     3721 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3781 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3841 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3901 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3961 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4021 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4081 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4141 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4201 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4261 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4321 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4381 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4441 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4501 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4561 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4621 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4681 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4741 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4801 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4861 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4921 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4981 ctggcctttt gctggccttt tgctcacatg tcctgcaggc agctgcgcgc tcgctcgctc
     5041 actgaggccg cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc
     5101 gcgcagagag ggagtggcca actccatcac taggggttcc tgcggccgca cgcgggaggg
     5161 cctattctgc ggacaaatgc tagaggtacc cgttaattaa tgcaaagatg gataaagttt
     5221 taaacagaga ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt
     5281 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
     5341 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
     5401 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
     5461 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
     5521 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
     5581 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
     5641 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
     5701 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
     5761 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
     5821 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
     5881 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
     5941 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
     6001 ggcc
//
