LOCUS       sequence-337025-        4962 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV vector expressing shRNA vs rat PKCd and a nuclear EYFP 
            reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4962)
  AUTHORS   Venniro M, Russell TI, Ramsey LA, Richie CT, Lesscher HMB, 
            Giovanetti SM, Messing RO, Shaham Y
  TITLE     Abstinence-dependent dissociable central amygdala microcircuits 
            control drug craving.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Apr 7;117(14):8126-8134. doi: 
            10.1073/pnas.2001615117. Epub 2020 Mar 23.
  PUBMED    32205443
REFERENCE   2  (bases 1 to 4962)
  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: 
            "10.1073/pnas.2001615117"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2020-04-7- 7"; volume: "117"; issue: "14"; pages: "8126-8134"
COMMENT     Sequence Label: pOTTC1534	pscAAV mU6 shRNA(PKCd) CMV-IE Nuc-EYFP
FEATURES             Location/Qualifiers
     source          1..4962
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(198..510)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(259..279)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     enhancer        605..908
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        909..1112
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1063..1083
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(1172..1174,1175..1177,1178..1888)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1217..1238)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1478..1497)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1825..1846
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1913..1933
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1937..1957
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1961..1981
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1996..2013)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2002..2113
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2225..2365
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2225..2354
                     /label=AAV2 ITR
     rep_origin      2440..2895
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2527..2546)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2737..2758
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2912..2931)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3031..3053
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3091..3109)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3177..3281
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3282..3350,3351..4142)
                     /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(3500..3519)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4313..4901
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4802..4821
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggctg
      121 cggccgcacg cgtcgacctc gagaaaaaag gacaaaggcc gcttcgaatc tcttgaattc
      181 gaagcggcct ttgtccaaac aaggcttttc tccaagggat atttatagtc tcaaaacaca
      241 caattacttt acagttaggg tgagtttcct tttgtgctgt tttttaaaat aataatttag
      301 tatttgtatc tcttatagaa atccaagcct atcatgtaaa atgtagctag tattaaaaag
      361 aacagattat ctgtctttta tcgcacatta agcctctata gttactagga aatattatat
      421 gcaaattaac cggggcaggg gagtagccga gcttctccca caagtctgtg cgagggggcc
      481 ggcgcgggcc tagagatggc gcgtcggatc tctagagcgg gcccggtaac gcgtggatct
      541 aatatagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
      601 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      661 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      721 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      781 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      841 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      901 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      961 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     1021 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     1081 gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgaagtt
     1141 atcgctagcg ctaccggact cagatctcac catggtgagc aagggcgagg agctgttcac
     1201 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1261 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1321 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcctgca
     1381 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1441 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1501 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1561 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1621 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1681 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1741 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc taccagtccg ccctgagcaa
     1801 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1861 cactctcggc atggacgagc tgtacaagtc cggcctgcgc agccgcgccg acccaaaaaa
     1921 gaagcgcaag gtagacccca agaagaagcg caaggtagat ccaaaaaaga agagaaaggt
     1981 atgatagtct agaagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2041 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2101 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2161 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggcg gaccgagcgg
     2221 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     2281 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     2341 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg
     2401 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     2461 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2521 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2581 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2641 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2701 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2761 caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     2821 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     2881 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     2941 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3001 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3061 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     3121 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     3181 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3241 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     3301 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     3361 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     3421 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     3481 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     3541 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     3601 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     3661 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     3721 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     3781 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     3841 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     3901 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     3961 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     4021 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     4081 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     4141 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     4201 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     4261 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4321 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4381 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4441 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4501 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4561 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4621 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4681 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4741 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4801 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4861 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4921 ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
