LOCUS       sequence-434457-        6473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses shRNA against CRH, marked with mCherry, in infected and 
            cre positive cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6473)
  AUTHORS   de Leon Reyes NS, Sierra Diaz P, Nogueira R, Ruiz-Pino A, Nomura Y, 
            de Solis CA, Schulkin J, Asok A, Leroy F
  TITLE     Corticotropin-releasing hormone signaling from prefrontal cortex to 
            lateral septum suppresses interaction with familiar mice.
  JOURNAL   Cell. 2023 Sep 14;186(19):4152-4171.e31. doi: 
            10.1016/j.cell.2023.08.010. Epub 2023 Sep 4.
  PUBMED    37669667
REFERENCE   2  (bases 1 to 6473)
  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: 
            "10.1016/j.cell.2023.08.010"; journalName: "Cell"; date: 
            "2023-09-14- 14"; volume: "186"; issue: "19"; pages: "4152-4171.e31"
COMMENT     Sequence Label: pAAV CMV-DIO-(mCherry-U6)-shRNA(anti-Crh)
FEATURES             Location/Qualifiers
     source          1..6473
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        206..509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        510..713
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     664..684
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     710..734
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          862..1240
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     protein_bind    complement(1317..1350)
                     /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(1401..1434)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(1439..1679)
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     primer_bind     complement(1489..1508)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1659..1679)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(1686..2396)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEYNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1964..1983)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2233..2251
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2393..2402
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2409..2442
                     /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    2493..2526
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2600..3188
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2653..2673)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3071..3082)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3220..3696
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3419..3438)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3736..3876
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3736..3865
                     /label=AAV2 ITR
     rep_origin      3951..4406
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4038..4057)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4248..4269
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4423..4442)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4542..4564
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4602..4620)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4688..4792
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4793..4861,4862..5653)
                     /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(5011..5030)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5824..6412
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6313..6332
                     /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 gcgtctagtt attaatagta atcaattacg gggtcattag
      181 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      241 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      301 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      361 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      421 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      481 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      541 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      601 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      661 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag
      721 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc
      781 gggaccgatc cagcctccgc ggattcgaat cccggccggg aacggtgcat tggaacgcgg
      841 attccccgtg ccaagagtga cgtaagtacc gcctatagag tctataggcc cacaaaaaat
      901 gctttcttct tttaatatac ttttttgttt atcttatttc taatactttc cctaatctct
      961 ttctttcagg gcaataatga tacaatgtat catgcctctt tgcaccattc taaagaataa
     1021 cagtgataat ttctgggtta aggcaatagc aatatttctg catataaata tttctgcata
     1081 taaattgtaa ctgatgtaag aggtttcata ttgctaatag cagctacaat ccagctacca
     1141 ttctgctttt attttatggt tgggataagg ctggattatt ctgagtccaa gctaggccct
     1201 tttgctaatc atgttcatac ctcttatctt cctcccacag ctcctgggca acgtgctggt
     1261 ctgtgtgctg gcccatcact ttggcaaaga attgggattc tagagtcgac tccggaataa
     1321 cttcgtatag gatactttat acgaagttat gcagaatggt agctggattg tagctgctat
     1381 tagcaatatg aaacctctta ataacttcgt atagcataca ttatacgaag ttattttcgt
     1441 cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac ggtaagcata
     1501 tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa ttattacttt
     1561 ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat tccaattatc
     1621 tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa taggccctcg
     1681 gatccttact tgtacagctc gtccatgccg ccggtggagt ggcggccctc ggcgcgttcg
     1741 tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt gatgttgacg
     1801 ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt gacctcagcg
     1861 tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt cagggcgccg
     1921 tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt ctgcattacg
     1981 gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc
     2041 tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcacgcgc
     2101 tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc
     2161 ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca ggcgaagggc
     2221 agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggcggccc
     2281 tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat gtgcaccttg
     2341 aagcgcatga actccttgat gatggccatg ttatactcct cgcccttgct caccatggtg
     2401 gcgctagcat aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat
     2461 tatcactgtt attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga
     2521 agttatcacc gggcatgggt gaagaatact tccttcaaga gaggaagtat tcttcaccca
     2581 tgctttttaa ttcactagta atcaacctct ggattacaaa atttgtgaaa gattgactgg
     2641 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     2701 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     2761 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     2821 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     2881 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     2941 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     3001 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     3061 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     3121 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     3181 ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc
     3241 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta
     3301 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg
     3361 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc
     3421 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc
     3481 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg
     3541 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg
     3601 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga
     3661 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc
     3721 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     3781 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     3841 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     3901 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     3961 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4021 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4081 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4141 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     4201 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4261 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
     4321 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4381 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     4441 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4501 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4561 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     4621 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     4681 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     4741 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     4801 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     4861 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     4921 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     4981 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5041 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5101 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5161 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5221 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5281 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5341 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     5401 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     5461 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     5521 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     5581 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     5641 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     5701 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     5761 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5821 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5881 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5941 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6001 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6061 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6121 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6181 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6241 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6301 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6361 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6421 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
