LOCUS       sequence-468744-        6027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express GFP from a CMV promoter and to express and shRNA 
            targeting Rat Npas4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6027)
  AUTHORS   Ploski JE, Monsey MS, Nguyen T, DiLeone RJ, Schafe GE
  TITLE     The neuronal PAS domain protein 4 (Npas4) is required for new and 
            reactivated fear memories.
  JOURNAL   PLoS One. 2011;6(8):e23760. doi: 10.1371/journal.pone.0023760. Epub 
            2011 Aug 22.
  PUBMED    21887312
REFERENCE   2  (bases 1 to 6027)
  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"; journalName: "PLoS One.";
            date: "2011"; volume: "6(8):e23760. doi"; pages: " 
            10.1371/journal.pone.0023760. Epub 2011 Aug 22"
COMMENT     Sequence Label: pAAV-CMV-GFP-pA U6 shRNA (Rat NPAS4 #1)
FEATURES             Location/Qualifiers
     source          1..6027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    37..513
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     295..314
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     CDS             complement(join(566..1279,1280..1282,1283..1285))
                     /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(569..1279,1280..1282,1283..1285))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(611..632)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     960..979
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1219..1240
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1282..1291
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          1391..1769
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     primer_bind     complement(1897..1921)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(1918..2121)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1947..1967)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2122..2425
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   2501..2630
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(2692..3280)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2772..2791)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3451..4242,4243..4311))
                     /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     4074..4093
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4312..4416)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4484..4502
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4540..4562)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4662..4681
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4698..5153)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4835..4856)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5047..5066
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5228..5368
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5239..5368
                     /label=AAV2 ITR
     polyA_signal    5482..5616
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5519..5538)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5573..5592
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(5695..6007)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(5756..5776)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
ORIGIN
        1 tggcgcgcca ttgggcccgg ttacctacaa aatcagaagg acagggaagg gagcagtggt
       61 tcacgcctgt aatcccagca atttgggagg ccaaggtggg tagatcacct gagattagga
      121 gttggagacc agcctggcca atatggtgaa accccgtctc taccaaaaaa acaaaaatta
      181 gctgagcctg gtcatgcatg cctggaatcc caacaactcg ggaggctgag gcaggagaat
      241 cgcttgaacc caggaggcgg agattgcagt gagccaagat tgtgccactg cactccagct
      301 tggttcccaa tagaccccgc aggccctaca ggttgtcttc ccaacttgcc ccttgctcca
      361 taccaccccc ctccacccca taatattata gaaggacacc tagtcagaca aaatgatgca
      421 acttaatttt attaggacaa ggctggtggg cactggagtg gcaacttcca gggccaggag
      481 aggcactggg gaggggtcac agggatgcca cccgtagatc tctcgagcag cgctgctcga
      541 ggcaagcttc tgcaggtcgg ccgctttact tgtacagctc gtccatgccg agagtgatcc
      601 cggcggcggt cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc
      661 tcagggcgga ctgggtgctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga
      721 tgggggtgtt ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc
      781 ggatcttgaa gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt
      841 ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga
      901 tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg
      961 tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca
     1021 tggcggactt gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg aagcactgca
     1081 cgccgtaggt cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc
     1141 agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc
     1201 tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga
     1261 acagctcctc gcccttgctc accatggtgg cgaccggtgg atccccgact gcagaattca
     1321 atcgatgttc gaatcccaat tctttgccaa agtgatgggc cagcacacag accagcacgt
     1381 tgcccaggag ctgtgggagg aagataagag gtatgaacat gattagcaaa agggcctagc
     1441 ttggactcag aataatccag ccttatccca accataaaat aaaagcagaa tggtagctgg
     1501 attgtagctg ctattagcaa tatgaaacct cttacatcag ttacaattta tatgcagaaa
     1561 tatttatatg cagaaatatt gctattgcct taacccagaa attatcactg ttattcttta
     1621 gaatggtgca aagaggcatg atacattgta tcattattgc cctgaaagaa agagattagg
     1681 gaaagtatta gaaataagat aaacaaaaaa gtatattaaa agaagaaagc attttttgtg
     1741 ggcctataga ctctataggc ggtacttacg tcactcttgg cacggggaat ccgcgttcca
     1801 atgcaccgtt cccggccggg attcgaatcc gcggaggctg gatcggtccc ggtgtcttct
     1861 atggaggtca aaacagcgtg gatggcgtct ccaggcgatc tgacggttca ctaaacgagc
     1921 tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca atggggcgga
     1981 gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac tcccattgac
     2041 gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac gcccattgat
     2101 gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga tgtactgcca
     2161 agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc
     2221 attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc
     2281 agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg gcgttactat
     2341 gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg
     2401 gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa ctaatgaccc
     2461 cgtaattgat tactattaat aactagacgc gtgcggccgc aggaacccct agtgatggag
     2521 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     2581 cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg acatgtgagc
     2641 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2701 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2761 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2821 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2881 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2941 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3001 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3061 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3121 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3181 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3241 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3301 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3361 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3421 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3481 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     3541 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     3601 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3661 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     3721 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     3781 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     3841 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     3901 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3961 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4021 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4081 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4141 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4201 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4261 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4321 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4381 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     4441 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     4501 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     4561 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     4621 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     4681 actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt
     4741 ttgttaaatc agctcatttt ttaaccaata gaccgaaatc ggcaaaatcc cttataaatc
     4801 aaaagaatag cccgagatag agttgagtgt tgttccagtt tggaacaaga gtccactatt
     4861 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     4921 acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     4981 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     5041 aaaggaaggg aagaaagcga aaggagcggg cgctaaggcg ctggcaagtg tagcggtcac
     5101 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtactatgg
     5161 ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     5221 aggcgcccct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
     5281 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
     5341 gtggccaact ccatcactag gggttcctgc ggccgctcgg tccgcacgtg gttaccggta
     5401 ccgccgcggc cgcaagaatt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     5461 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     5521 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5581 tccaaactca tcaatgtatc ttatcatgtc tggatctacg tattatctag aaaaattgaa
     5641 ctggttagtg gatctgggat gacaggaagt cccagatcca ctaaccagtt cacaaacaag
     5701 gcttttctcc aagggatatt tatagtctca aaacacacaa ttactttaca gttagggtga
     5761 gtttcctttt gtgctgtttt ttaaaataat aatttagtat ttgtatctct tatagaaatc
     5821 caagcctatc atgtaaaatg tagctagtat taaaaagaac agattatctg tcttttatcg
     5881 cacattaagc ctctatagtt actaggaaat attatatgca aattaaccgg ggcaggggag
     5941 tagccgagct tctcccacaa gtctgtgcga gggggccggc gcgggcctag agatggcggc
     6001 gtcggataag cttggcctag atggcct
//
