LOCUS       sequence-260226-        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 Pdyn gene, which encodes 
            dynorphin.
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-shDyn
FEATURES             Location/Qualifiers
     source          1..5937
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        149..596
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(634..667)
                     /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(718..751)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     ncRNA           complement(788..914)
                     /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(942..956)
                     /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(984..1105)
                     /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(1154..1867,1868..1870,1871..1873))
                     /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(1157..1867,1868..1870,1871..1873))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1199..1220)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1548..1567
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1807..1828
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1870..1879
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1896..1929
                     /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    1980..2013
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2038..2626
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2091..2111)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2509..2520)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2658..3134
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2857..2876)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3174..3314
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3174..3303
                     /label=AAV2 ITR
     rep_origin      3389..3844
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3476..3495)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3686..3707
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3861..3880)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3980..4002
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4040..4058)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4126..4230
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4231..4299,4300..5091)
                     /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(4449..4468)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5262..5850
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5751..5770
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(5912..5937,1..104)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg
       61 agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc gcacgcgtgt
      121 gtctagactg cagagggccc tgcgtatgag tgcaagtggg ttttaggacc aggatgaggc
      181 ggggtggggg tgcctacctg acgaccgacc ccgacccact ggacaagcac ccaaccccca
      241 ttccccaaat tgcgcatccc ctatcagaga gggggagggg aaacaggatg cggcgaggcg
      301 cgtgcgcact gccagcttca gcaccgcgga cagtgccttc gcccccgcct ggcggcgcgc
      361 gccaccgccg cctcagcact gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc
      421 cccttcccgg ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg
      481 cgaggcgcga gatagggggg cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag
      541 cgctgcctca gtctgcggtg ggcagcggag gagtcgtgtc gtgcctgaga gcgcagtcga
      601 gaaggtaccg gatcctctag agtcgactcc ggaataactt cgtataggat actttatacg
      661 aagttatgca gaatggtagc tggattgtag ctgctattag caatatgaaa cctcttaata
      721 acttcgtata gcatacatta tacgaagtta tggcgcgcct ttaaacgcat tagtcttcca
      781 attgaaaaaa gtgatttaat ttataccatt ttaattcagc tttgtaaaaa tgtatcaaag
      841 agatagcaag gtattcagtt ttagtaaaca agataattgc tcctaaagta gccccttgaa
      901 ttccgaggca gtaggcatac actgagcctc gttctccatt acatctgtgg cttcactaat
      961 ggagaacgag gctcagtgtg cgctcactgt caacagcaat ataccttctc gagccttctg
     1021 ttgggttaac ctgaagaagt aatcccagca agtgtttcca agatgtgcag gcaacgattc
     1081 tgtaaagtac tgaagcctca ttcaacatag tatatgtgct gccgaagcga gcacttaaca
     1141 aggcttgcgg ccgctacttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca
     1201 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact
     1261 gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
     1321 gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt
     1381 tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt
     1441 tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct
     1501 cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc
     1561 cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga
     1621 agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca
     1681 gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca
     1741 gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc
     1801 cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc
     1861 ccttgctcac catggtggcg accggtagcg ctagcataac ttcgtataaa gtatcctata
     1921 cgaagttatt tgccttaacc cagaaattat cactgttatt ctttagaatg gtgcaaagaa
     1981 taacttcgta taatgtatgc tatacgaagt tatgaattcg atatcaagct tatcgataat
     2041 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     2101 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     2161 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     2221 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     2281 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     2341 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     2401 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     2461 tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     2521 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     2581 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgagcg
     2641 ctgctcgaga gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc
     2701 tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt
     2761 tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg
     2821 ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg
     2881 cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc
     2941 ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt
     3001 ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc
     3061 aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc
     3121 ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc
     3181 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     3241 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     3301 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3361 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     3421 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     3481 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3541 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3601 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3661 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3721 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     3781 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     3841 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     3901 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3961 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4021 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     4081 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4141 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4201 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4261 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4321 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4381 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4441 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4501 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4561 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4621 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4681 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4741 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4801 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4861 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4921 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4981 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5041 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5101 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5161 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5221 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5281 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5341 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5401 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5461 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5521 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5581 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5641 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5701 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5761 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5821 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5881 ctggcctttt gctggccttt tgctcacatg tcctgcaggc agctgcgcgc tcgctcg
//
