LOCUS       sequence-337032-        5941 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV vector expressing miR-30a shRNA vs FF luciferase and a 
            nuclear EYFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5941)
  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 5941)
  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: pOTTC1618	pAAV SYN1 Nuc-EYFP miR-30 FF3
FEATURES             Location/Qualifiers
     source          1..5941
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        186..633
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             join(690..692,693..695,696..1406)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(735..756)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(996..1015)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1343..1364
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1431..1451
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1455..1475
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1479..1499
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     ncRNA           1569..1690
                     /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"
     ncRNA           1717..1731
                     /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           1759..1885
                     /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"
     misc_feature    2068..2656
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2121..2141)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2539..2550)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2688..3164
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2887..2906)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3204..3344
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3204..3333
                     /label=AAV2 ITR
     rep_origin      3419..3874
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3506..3525)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3716..3737
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3891..3910)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4010..4032
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4070..4088)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4156..4260
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4261..4329,4330..5121)
                     /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(4479..4498)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5292..5880
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5781..5800
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtgtgtc tagactgcag agggccctgc
      181 gtatgagtgc aagtgggttt taggaccagg atgaggcggg gtgggggtgc ctacctgacg
      241 accgaccccg acccactgga caagcaccca acccccattc cccaaattgc gcatccccta
      301 tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca
      361 ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa
      421 ggcgcgctga cgtcactcgc cggtcccccg caaactcccc ttcccggcca ccttggtcgc
      481 gtccgcgccg ccgccggccc agccggaccg caccacgcga ggcgcgagat aggggggcac
      541 gggcgcgacc atctgcgctg cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc
      601 agcggaggag tcgtgtcgtg cctgagagcg cagtcgagaa ggtaccggat ccgaagttat
      661 cgctagcgct accggactca gatctcacca tggtgagcaa gggcgaggag ctgttcaccg
      721 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
      781 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
      841 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcctgcagt
      901 gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
      961 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1021 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1081 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     1141 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     1201 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1261 acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccgcc ctgagcaaag
     1321 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1381 ctctcggcat ggacgagctg tacaagtccg gcctgcgcag ccgcgccgac ccaaaaaaga
     1441 agcgcaaggt agaccccaag aagaagcgca aggtagatcc aaaaaagaag agaaaggtat
     1501 gataggcgcg ccataacttc cggccgcaag ccttgttaag tgctcgcttc ggcagcacat
     1561 atactatgtt gaatgaggct tcagtacttt acagaatcgt tgcctgcaca tcttggaaac
     1621 acttgctggg attacttctt caggttaacc caacagaagg ctcgagaagg tatattgctg
     1681 ttgacagtga gcgagctccc gtgaattgga atcctagtga agccacagat gtaggattcc
     1741 aattcagcgg gagcctgcct actgcctcgg aattcaaggg gctactttag gagcaattat
     1801 cttgtttact aaaactgaat accttgctat ctctttgata catttttaca aagctgaatt
     1861 aaaatggtat aaattaaatc acttttttca attggaagac taatgcgttt aaacacgcgg
     1921 cgacgcgttc gaccgaataa atacctgtga cggaagatca cttcgcagaa taaataaatc
     1981 ctggtgtccc tgttgatacc gggaagccct gggccaactt ttggcgaaaa tgagacgttg
     2041 atcgaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga
     2101 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     2161 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     2221 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     2281 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     2341 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     2401 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     2461 aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg
     2521 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     2581 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     2641 tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga gatctacggg tggcatccct
     2701 gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc
     2761 ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta
     2821 tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct
     2881 gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct
     2941 ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg
     3001 catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat
     3061 tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat
     3121 tgctgggatt acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa
     3181 ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     3241 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     3301 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     3361 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     3421 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3481 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3541 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3601 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     3661 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3721 ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa
     3781 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     3841 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     3901 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     3961 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4021 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     4081 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     4141 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4201 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4261 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     4321 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4381 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     4441 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     4501 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     4561 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     4621 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     4681 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     4741 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     4801 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     4861 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     4921 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4981 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5041 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     5101 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     5161 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5221 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5281 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5341 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5401 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     5461 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5521 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5581 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5641 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5701 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5761 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5821 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5881 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5941 t
//
