LOCUS       sequence-341670-        5930 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 5930)
  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 5930)
  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..5930
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        174..621
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             join(678..680,681..683,684..1394)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(723..744)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(984..1003)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1331..1352
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1419..1439
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1443..1463
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1467..1487
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     ncRNA           1557..1678
                     /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           1705..1719
                     /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           1747..1873
                     /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    2056..2644
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2109..2129)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2527..2538)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2676..3152
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2875..2894)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3192..3332
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3192..3321
                     /label=AAV2 ITR
     rep_origin      3407..3862
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3494..3513)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3704..3725
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3879..3898)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3998..4020
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4058..4076)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4144..4248
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4249..4317,4318..5109)
                     /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(4467..4486)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5280..5868
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5769..5788
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(5930,1..129)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg
       61 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
      121 ggggttcctg cggccgcacg cgtgtgtcta gactgcagag ggccctgcgt atgagtgcaa
      181 gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac
      241 ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg
      301 aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg
      361 ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg
      421 tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc
      481 gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat
      541 ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc
      601 gtgtcgtgcc tgagagcgca gtcgagaagg taccggatcc gaagttatcg ctagcgctac
      661 cggactcaga tctcaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      721 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
      781 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      841 ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cctgcagtgc ttcgcccgct
      901 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
      961 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1021 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1081 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1141 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     1201 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1261 tgctgcccga caaccactac ctgagctacc agtccgccct gagcaaagac cccaacgaga
     1321 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1381 acgagctgta caagtccggc ctgcgcagcc gcgccgaccc aaaaaagaag cgcaaggtag
     1441 accccaagaa gaagcgcaag gtagatccaa aaaagaagag aaaggtatga taggcgcgcc
     1501 ataacttccg gccgcaagcc ttgttaagtg ctcgcttcgg cagcacatat actatgttga
     1561 atgaggcttc agtactttac agaatcgttg cctgcacatc ttggaaacac ttgctgggat
     1621 tacttcttca ggttaaccca acagaaggct cgagaaggta tattgctgtt gacagtgagc
     1681 gagctcccgt gaattggaat cctagtgaag ccacagatgt aggattccaa ttcagcggga
     1741 gcctgcctac tgcctcggaa ttcaaggggc tactttagga gcaattatct tgtttactaa
     1801 aactgaatac cttgctatct ctttgataca tttttacaaa gctgaattaa aatggtataa
     1861 attaaatcac ttttttcaat tggaagacta atgcgtttaa acacgcggcg acgcgttcga
     1921 ccgaataaat acctgtgacg gaagatcact tcgcagaata aataaatcct ggtgtccctg
     1981 ttgataccgg gaagccctgg gccaactttt ggcgaaaatg agacgttgat cgaattcgat
     2041 atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     2101 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     2161 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     2221 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     2281 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     2341 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     2401 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     2461 tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac
     2521 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     2581 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     2641 ccgcatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt gacccctccc
     2701 cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa
     2761 aattaagttg catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg
     2821 gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt
     2881 gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg
     2941 ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca
     3001 ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg
     3061 tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac
     3121 aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac
     3181 cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     3241 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     3301 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     3361 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     3421 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3481 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3541 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3601 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     3661 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3721 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     3781 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     3841 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     3901 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3961 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4021 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     4081 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     4141 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4201 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4261 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     4321 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     4381 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     4441 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     4501 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     4561 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     4621 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     4681 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     4741 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     4801 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     4861 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     4921 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     4981 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5041 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5101 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5161 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5221 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5281 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5341 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5401 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5461 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5521 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     5581 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     5641 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5701 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5761 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5821 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5881 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtc
//
