LOCUS       sequence-417791-        6001 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV genome to express mCherry 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 6001)
  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 6001)
  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: pAS132
FEATURES             Location/Qualifiers
     source          1..6001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     572..591
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(810..914)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     982..1000
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1038..1060)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1160..1179
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(1196..1651)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1333..1354)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1545..1564
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   1726..1866
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1737..1866
                     /label=AAV2 ITR
     polyA_signal    1890..2024
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1914..1933)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1968..1987
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2174..2185
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2583..2603
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2954..3661)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3229..3248)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3498..3516
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3658..3667
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3682..4860)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3691..4629
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3714..3734)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     repeat_region   5000..5129
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(5191..5779)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5271..5290)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5950..6001,1..740,741..809))
                     /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"
ORIGIN
        1 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
       61 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
      121 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
      181 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
      241 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
      301 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
      361 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
      421 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
      481 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
      541 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
      601 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
      661 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
      721 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
      781 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
      841 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
      901 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
      961 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtat cgcgcgtttc
     1021 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agaaggtcac agcttgtctg
     1081 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     1141 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccataaaatt
     1201 gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     1261 aaccaataga ccgaaatcgg caaaatccct tataaatcaa aagaatagcc cgagatagag
     1321 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     1381 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc acccaaatca
     1441 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     1501 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     1561 ggagcgggcg ctaaggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     1621 gccgcgctta atgcgccgct acagggcgcg tactatggtt gctttgacgt atgcggtgtg
     1681 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgcccctgc aggcagctgc
     1741 gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg acctttggtc
     1801 gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg
     1861 gttcctgcgg ccgctcggtc cgcacgtgag atccagacat gataagatac attgatgagt
     1921 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     1981 ctattgcttt atttgtaacc attataagct gcaataaaca agttatcgat tgcattctag
     2041 catttgtctg cagaataggc cctcccgcgt gggaggcggc ccaaagggag atccgactcg
     2101 tctgagggcg aaggcgaaga cgcggaagag gccgcagagc cggcagcagg ccgcgggaag
     2161 gaaggtccgc tggattgagg gccgaaggga cgtagcagaa ggacgtcccg cgcagaatcc
     2221 aggtggcaac ataggcgagc agccaaggaa aggacgatga tttccccgac aacaccacgg
     2281 aattgtcagt gcccaacagc cgagcccctg tccagcagcg ggcaaggcag gcggcgatga
     2341 gttccgccgt ggcaataggg agggggaaag cgaaagtccc ggaaaggagc tgacaggtgg
     2401 tggcaatgcc ccaaccagtg ggggttgcgt cagcaaacac agtgcacacc acgccacgtt
     2461 gcctgacaac gggccacaac tcctcataaa gagacagcaa ccaggattta tacaaggagg
     2521 agaaaatgaa agccatacgg gaagcaatag catgatacaa aggcattaaa gcagcgtatc
     2581 cacatagcgt aaaaggagca acatagttaa gaataccagt caatctttca caaattttgt
     2641 aatccagagg ttgattatcg ataagcttga tatcgaattc ctaggcttag gtgggaggtg
     2701 gcccgttggt gtaccgcagc atggggtaga aggatcgggc aaagttgttg gcctgggtgc
     2761 agctgtagtc atcttcagag gcaggtagca ggcaggccag gagcagcagc agcagcagaa
     2821 gcagctgcaa cggtagcgct gccctgatca cccttgagag gaaggagcgt ctaggctggg
     2881 agctgccggg gctgtcgagg tggggcatcc tgctgtctgt ctcctcctcc tcctctcgag
     2941 atctgagtcc ggacttgtac agctcgtcca tgccgccggt ggagtggcgg ccctcggcgc
     3001 gttcgtactg ttccacgatg gtgtagtcct cgttgtggga ggtgatgtcc aacttgatgt
     3061 tgacgttgta ggcgccgggc agctgcacgg gcttcttggc cttgtaggtg gtcttgacct
     3121 cagcgtcgta gtggccgccg tccttcagct tcagcctctg cttgatctcg cccttcaggg
     3181 cgccgtcctc ggggtacatc cgctcggagg aggcctccca gcccatggtc ttcttctgca
     3241 ttacggggcc gtcggagggg aagttggtgc cgcgcagctt caccttgtag atgaactcgc
     3301 cgtcctgcag ggaggagtcc tgggtcacgg tcaccacgcc gccgtcctcg aagttcatca
     3361 cgcgctccca cttgaagccc tcggggaagg acagcttcaa gtagtcgggg atgtcggcgg
     3421 ggtgcttcac gtaggccttg gagccgtaca tgaactgagg ggacaggatg tcccaggcga
     3481 agggcagggg gccacccttg gtcaccttca gcttggcggt ctgggtgccc tcgtaggggc
     3541 ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt cacggagccc tccatgtgca
     3601 ccttgaagcg catgaactcc ttgatgatgg ccatgttatc ctcctcgccc ttgctcacca
     3661 tggtggcacc ggtggcgcgc ctcacgacac ctgaaatgga agaaaaaaac tttgaaccac
     3721 tgtctgaggc ttgagaatga accaagatcc aaactcaaaa agggcaaatt ccaaggagaa
     3781 ttacatcaag tgccaagctg gcctaacttc agtctccacc cactcagtgt ggggaaactc
     3841 catcgcataa aacccctccc cccaacctaa agacgacgta ctccaaaagc tcgagaacta
     3901 atcgaggtgc ctggacggcg cccggtactc cgtggagtca catgaagcga cggctgagga
     3961 cggaaaggcc cttttccttt gtgtgggtga ctcacccgcc cgctctcccg agcgccgcgt
     4021 cctccatttt gagctccctg cagcagggcc gggaagcggc catctttccg ctcacgcaac
     4081 tggtgccgac cgggccagcc ttgccgccca gggcggggcg atacacggcg gcgcgaggcc
     4141 aggcaccaga gcaggccggc cagcttgaga ctacccccgt ccgattctcg gtggccgcgc
     4201 tcgcaggccc cgcctcgccg aacatgtgcg ctgggacgca cgggccccgt cgccgcccgc
     4261 ggccccaaaa accgaaatac cagtgtgcag atcttggccc gcatttacaa gactatcttg
     4321 ccagaaaaaa agcgtcgcag caggtcatca aaaattttaa atggctagag acttatcgaa
     4381 agcagcgaga caggcgcgaa ggtgccacca gattcgcacg cggcggcccc agcgcccagg
     4441 ccaggcctca actcaagcac gaggcgaagg ggctccttaa gcgcaaggcc tcgaactctc
     4501 ccacccactt ccaacccgaa gctcgggatc aagaatcacg tactgcagcc aggtggaagt
     4561 aattcaaggc acgcaagggc cataacccgt aaagaggcca ggcccgcggg aaccacacac
     4621 ggcacttacc tgtgttctgg cggcaaaccc gttgcgaaaa agaacgttca cggcgactac
     4681 tgcacttata tacggttctc ccccaccctc gggaaaaagg cggagccagt acacgacatc
     4741 actttcccag tttaccccgc gccaccttct ctaggcaccg gttcaattgc cgacccctcc
     4801 ccccaacttc tcggggactg tgggcgatgt gcgctctgcc cactgacggg caccggagcc
     4861 aattcccact cctttcaaga cctagaaggt ccattagctg caaagattcc tctctgttta
     4921 aaactttatc catctttgca ttaattaacg ggtacctcta gcatttgtcc gcagaatagg
     4981 ccctcccgcg tgcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     5041 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg cggcctcagt
     5101 gagcgagcga gcgcgcagct gcctgcagga catgtgagca aaaggccagc aaaaggccag
     5161 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5221 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5281 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5341 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5401 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5461 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5521 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5581 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5641 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5701 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5761 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5821 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5881 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5941 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6001 t
//
