LOCUS       sequence-468747-        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 #5)
FEATURES             Location/Qualifiers
     source          1..6027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        127..330
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     281..301
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     327..351
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          479..857
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     regulatory      957..966
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(963..965,966..968,969..1682)
                     /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             join(963..965,966..968,969..1679)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1008..1029)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1269..1288)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1616..1637
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1735..2211
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1934..1953)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     promoter        2268..2580
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2499..2519
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     polyA_signal    2659..2793
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2683..2702)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2737..2756
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   2907..3047
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2907..3036
                     /label=AAV2 ITR
     rep_origin      3122..3577
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3209..3228)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3419..3440
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3594..3613)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3713..3735
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3773..3791)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3859..3963
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3964..4032,4033..4824)
                     /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(4182..4201)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4995..5583
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5484..5503
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5645..5774
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        join(5850..6027,1..126)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
       61 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      121 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      181 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      241 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      301 gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga
      361 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccgcgga
      421 ttcgaatccc ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca agagtgacgt
      481 aagtaccgcc tatagagtct ataggcccac aaaaaatgct ttcttctttt aatatacttt
      541 tttgtttatc ttatttctaa tactttccct aatctctttc tttcagggca ataatgatac
      601 aatgtatcat gcctctttgc accattctaa agaataacag tgataatttc tgggttaagg
      661 caatagcaat atttctgcat ataaatattt ctgcatataa attgtaactg atgtaagagg
      721 tttcatattg ctaatagcag ctacaatcca gctaccattc tgcttttatt ttatggttgg
      781 gataaggctg gattattctg agtccaagct aggccctttt gctaatcatg ttcatacctc
      841 ttatcttcct cccacagctc ctgggcaacg tgctggtctg tgtgctggcc catcactttg
      901 gcaaagaatt gggattcgaa catcgattga attctgcagt cggggatcca ccggtcgcca
      961 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1021 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1081 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1141 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1201 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1261 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1321 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1381 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1441 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1501 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1561 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1621 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     1681 aaagcggccg acctgcagaa gcttgcctcg agcagcgctg ctcgagagat ctacgggtgg
     1741 catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc
     1801 accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat
     1861 aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt
     1921 agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact
     1981 gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga
     2041 ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc
     2101 accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct
     2161 cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgattttg
     2221 taggtaaccg ggcccaatgg cgcgccaagg ccatctaggc caagcttatc cgacgccgcc
     2281 atctctaggc ccgcgccggc cccctcgcac agacttgtgg gagaagctcg gctactcccc
     2341 tgccccggtt aatttgcata taatatttcc tagtaactat agaggcttaa tgtgcgataa
     2401 aagacagata atctgttctt tttaatacta gctacatttt acatgatagg cttggatttc
     2461 tataagagat acaaatacta aattattatt ttaaaaaaca gcacaaaagg aaactcaccc
     2521 taactgtaaa gtaattgtgt gttttgagac tataaatatc ccttggagaa aagccttgtt
     2581 tgatctgctg gatgagacga tctatcttcc tgtcaataga tcgtctcatc cagcagatat
     2641 ttttctagat aatacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc
     2701 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     2761 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     2821 tttcaggttc agggggaggt gtgggaggtt ttttaattct tgcggccgcg gcggtaccgg
     2881 taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     2941 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     3001 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     3061 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     3121 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3181 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3241 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3301 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     3361 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3421 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     3481 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     3541 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     3601 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     3661 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3721 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     3781 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     3841 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3901 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3961 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4021 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4081 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4141 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4201 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4261 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4321 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4381 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4441 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4501 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4561 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4621 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4681 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4741 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4801 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4861 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4921 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4981 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5041 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5101 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     5161 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5221 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5281 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5341 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5401 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5461 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5521 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5581 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5641 atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac
     5701 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
     5761 cactaggggt tcctgcggcc gcacgcgtct agttattaat agtaatcaat tacggggtca
     5821 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     5881 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     5941 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     6001 ttggcagtac atcaagtgta tcatatg
//
