LOCUS       sequence-260225-        5937 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cre-dependent short hairpin RNA (shRNA) that targets the 
            3'-untranslated region of the rat Crh gene which encodes 
            corticotropin releasing factor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5937)
  AUTHORS   Pomrenze MB, Giovanetti SM, Maiya R, Gordon AG, Kreeger LJ, Messing 
            RO
  TITLE     Dissecting the Roles of GABA and Neuropeptides from Rat Central 
            Amygdala CRF Neurons in Anxiety and Fear Learning.
  JOURNAL   Cell Rep. 2019 Oct 1;29(1):13-21.e4. doi: 
            10.1016/j.celrep.2019.08.083.
  PUBMED    31577943
REFERENCE   2  (bases 1 to 5937)
  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.celrep.2019.08"; journalName: "Cell Rep"; date: 
            "2019-10-1- 1"; volume: "29"; issue: "1"; pages: "13-21.e4"
COMMENT     Sequence Label: AAV8-hSyn-flex-miR30-eGFP-shCrh
FEATURES             Location/Qualifiers
     source          1..5937
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   39..168
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        213..660
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(698..731)
                     /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(782..815)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     ncRNA           complement(852..978)
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(1006..1020)
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(1048..1169)
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     CDS             complement(join(1218..1931,1932..1934,1935..1937))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1221..1931,1932..1934,1935..1937))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1263..1284)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1612..1631
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1871..1892
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1934..1943
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1960..1993
                     /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    2044..2077
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2102..2690
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2155..2175)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2573..2584)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2722..3198
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2921..2940)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3238..3378
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3238..3367
                     /label=AAV2 ITR
     rep_origin      3453..3908
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3540..3559)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3750..3771
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3925..3944)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4044..4066
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4104..4122)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4190..4294
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4295..4363,4364..5155)
                     /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(4513..4532)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5326..5914
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5815..5834
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc tgcgcgctcg
       61 ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
      121 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc ggccgcacgc
      181 gtgtgtctag actgcagagg gccctgcgta tgagtgcaag tgggttttag gaccaggatg
      241 aggcggggtg ggggtgccta cctgacgacc gaccccgacc cactggacaa gcacccaacc
      301 cccattcccc aaattgcgca tcccctatca gagaggggga ggggaaacag gatgcggcga
      361 ggcgcgtgcg cactgccagc ttcagcaccg cggacagtgc cttcgccccc gcctggcggc
      421 gcgcgccacc gccgcctcag cactgaaggc gcgctgacgt cactcgccgg tcccccgcaa
      481 actccccttc ccggccacct tggtcgcgtc cgcgccgccg ccggcccagc cggaccgcac
      541 cacgcgaggc gcgagatagg ggggcacggg cgcgaccatc tgcgctgcgg cgccggcgac
      601 tcagcgctgc ctcagtctgc ggtgggcagc ggaggagtcg tgtcgtgcct gagagcgcag
      661 tcgagaaggt accggatcct ctagagtcga ctccggaata acttcgtata ggatacttta
      721 tacgaagtta tgcagaatgg tagctggatt gtagctgcta ttagcaatat gaaacctctt
      781 aataacttcg tatagcatac attatacgaa gttatggcgc gcctttaaac gcattagtct
      841 tccaattgaa aaaagtgatt taatttatac cattttaatt cagctttgta aaaatgtatc
      901 aaagagatag caaggtattc agttttagta aacaagataa ttgctcctaa agtagcccct
      961 tgaattccga ggcagtaggc aaacacagta ttctgtacca tactacatct gtggcttcac
     1021 tagtatggta cagaatactg tgtgcgctca ctgtcaacag caatatacct tctcgagcct
     1081 tctgttgggt taacctgaag aagtaatccc agcaagtgtt tccaagatgt gcaggcaacg
     1141 attctgtaaa gtactgaagc ctcattcaac atagtatatg tgctgccgaa gcgagcactt
     1201 aacaaggctt gcggccgcta cttgtacagc tcgtccatgc cgagagtgat cccggcggcg
     1261 gtcacgaact ccagcaggac catgtgatcg cgcttctcgt tggggtcttt gctcagggcg
     1321 gactgggtgc tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg
     1381 ttctgctggt agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg
     1441 aagttcacct tgatgccgtt cttctgcttg tcggccatga tatagacgtt gtggctgttg
     1501 tagttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc
     1561 agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag
     1621 ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt agccttcggg catggcggac
     1681 ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc tgaagcactg cacgccgtag
     1741 gtcagggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt gcagatgaac
     1801 ttcagggtca gcttgccgta ggtggcatcg ccctcgccct cgccggacac gctgaacttg
     1861 tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt gaacagctcc
     1921 tcgcccttgc tcaccatggt ggcgaccggt agcgctagca taacttcgta taaagtatcc
     1981 tatacgaagt tatttgcctt aacccagaaa ttatcactgt tattctttag aatggtgcaa
     2041 agaataactt cgtataatgt atgctatacg aagttatgaa ttcgatatca agcttatcga
     2101 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     2161 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     2221 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     2281 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     2341 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     2401 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     2461 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     2521 cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     2581 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     2641 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     2701 agcgctgctc gagagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg
     2761 gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc
     2821 attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc
     2881 aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg
     2941 agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc
     3001 ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt
     3061 ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa
     3121 tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact
     3181 gctcccttcc ctgtccttct gattttgtag gtaaccacgt gcggaccgag cggccgcagg
     3241 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     3301 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     3361 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3421 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     3481 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     3541 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3601 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3661 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3721 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3781 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     3841 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     3901 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     3961 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4021 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4081 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     4141 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4201 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4261 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     4321 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     4381 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     4441 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     4501 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     4561 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     4621 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     4681 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     4741 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     4801 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     4861 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     4921 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     4981 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     5041 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     5101 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     5161 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5221 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     5281 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5341 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5401 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5461 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5521 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5581 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5641 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     5701 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5761 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5821 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5881 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg ccttttt
//
