LOCUS       sequence-265127-        6751 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone that can be used to generate AAV virus where, upon 
            cre-specific recombination, mCherry and any shRNA can be 
            simultaneously expressed.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6751)
  AUTHORS   Favuzzi E, Deogracias R, Marques-Smith A, Maeso P, Jezequel J, 
            Exposito-Alonso D, Balia M, Kroon T, Hinojosa AJ, F Maraver E, Rico 
            B
  TITLE     Distinct molecular programs regulate synapse specificity in cortical
            inhibitory circuits.
  JOURNAL   Science. 2019 Jan 25;363(6425):413-417. doi: 
            10.1126/science.aau8977.
  PUBMED    30679375
REFERENCE   2  (bases 1 to 6751)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2019-01-25- 25"; volume: "363"; 
            issue: "6425"; pages: "413-417"
COMMENT     Sequence Label: pDIO-DSE-mCherry-PSE-MCS
FEATURES             Location/Qualifiers
     source          1..6751
                     /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        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1446..1479)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1530..1563)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1652..1672)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(1772..2482)
                     /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(2050..2069)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2319..2337
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2479..2488
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2610..2643
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    2694..2727
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2763..2782
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2878..3466
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2931..2951)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3349..3360)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3498..3974
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3697..3716)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4014..4154
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4014..4143
                     /label=AAV2 ITR
     rep_origin      4229..4684
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4316..4335)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4526..4547
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4701..4720)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4820..4842
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4880..4898)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4966..5070
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5071..5139,5140..5931)
                     /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(5289..5308)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6102..6690
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6591..6610
                     /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 gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagattc gctagcggat
     1441 ccggaataac ttcgtatagg atactttata cgaagttatg cagaatggta gctggattgt
     1501 agctgctatt agcaatatga aacctcttaa taacttcgta tagcatacat tatacgaagt
     1561 tattattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt atctctctaa
     1621 cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc tcttcctgcc
     1681 cgaccttggc gcgcgctcgg cgcgcggtca cgctccgtca cgtggtgcgt tttgcctgcg
     1741 cgtctttcca ctgggttaat taagcggccg cttacttgta cagctcgtcc atgccgccgg
     1801 tggagtggcg gccctcggcg cgttcgtact gttccacgat ggtgtagtcc tcgttgtggg
     1861 aggtgatgtc caacttgatg ttgacgttgt aggcgccggg cagctgcacg ggcttcttgg
     1921 ccttgtaggt ggtcttgacc tcagcgtcgt agtggccgcc gtccttcagc ttcagcctct
     1981 gcttgatctc gcccttcagg gcgccgtcct cggggtacat ccgctcggag gaggcctccc
     2041 agcccatggt cttcttctgc attacggggc cgtcggaggg gaagttggtg ccgcgcagct
     2101 tcaccttgta gatgaactcg ccgtcctgca gggaggagtc ctgggtcacg gtcaccacgc
     2161 cgccgtcctc gaagttcatc acgcgctccc acttgaagcc ctcggggaag gacagcttca
     2221 agtagtcggg gatgtcggcg gggtgcttca cgtaggcctt ggagccgtac atgaactgag
     2281 gggacaggat gtcccaggcg aagggcaggg ggccaccctt ggtcaccttc agcttggcgg
     2341 tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc gatctcgaac tcgtggccgt
     2401 tcacggagcc ctccatgtgc accttgaagc gcatgaactc cttgatgatg gccatgttat
     2461 cctcctcgcc cttgctcacc atggtggcgg atgctaccgg tggatcccgg gcccgcggta
     2521 ccgtcgactg cagctcctga tcggcctctt cattcttctc aaccggttta gttcttctga
     2581 tgcagcacag catggggcgg cgcgccgcga taacttcgta taaagtatcc tatacgaagt
     2641 tatttgcctt aacccagaaa ttatcactgt tattctttag aatggtgcaa agaataactt
     2701 cgtataatgt atgctatacg aagttatgtt tatctgcagt tttaaaatta tgttttaaaa
     2761 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttgggt ttatatatct
     2821 tgtggaaagg acgcggtttc ctaggtttaa actgaattcg atatcaagct tatcgataat
     2881 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     2941 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     3001 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     3061 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     3121 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     3181 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3241 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     3301 tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3361 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3421 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgagcg
     3481 ctgctcgaga gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc
     3541 tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt
     3601 tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg
     3661 ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg
     3721 cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc
     3781 ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt
     3841 ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc
     3901 aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc
     3961 ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc
     4021 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     4081 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     4141 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4201 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     4261 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     4321 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4381 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4441 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4501 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4561 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     4621 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     4681 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     4741 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4801 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4861 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     4921 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4981 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5041 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5101 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5161 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5221 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5281 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5341 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5401 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5461 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5521 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5581 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     5641 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5701 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5761 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5821 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5881 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5941 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6001 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6061 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6121 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6181 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6241 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6301 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6361 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6421 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6481 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6541 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6601 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6661 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6721 ctggcctttt gctggccttt tgctcacatg t
//
