LOCUS       Exported                8637 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Encodes short hairpin RNA (shRNA) #1 that targets the 
            3'-untranslated region of the rat Pdyn gene.
ACCESSION   .
VERSION     .
KEYWORDS    pPRIME-CMV-GFP-shDyn1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8637)
  AUTHORS   Pomrenze MB, Giovanetti SM, Maiya R, Gordon AG, Kreeger LJ, Messing 
            RO
  TITLE     Dissecting the Roles of GABA and Neuropeptides from Rat Central 
            Amygdala CRF Neurons in Anxiety and Fear Learning.
  JOURNAL   Cell Rep. 2019 Oct 1;29(1):13-21.e4. doi: 
            10.1016/j.celrep.2019.08.083.
  PUBMED    31577943
REFERENCE   2  (bases 1 to 8637)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8637
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    110..227
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        364..667
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        668..871
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      910..919
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             916..1632
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(961..982)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1222..1241)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1569..1590
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           1684..1805
                     /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           1833..1847
                     /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           1875..2001
                     /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"
     promoter        2155..2257
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             2258..2917
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2324..2343)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3047..3076
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     3101..3117
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3192..3780
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3245..3265)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3663..3674)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3783..3799)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             4309..4489
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4551..5139)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4631..4650)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5310..6170)
                     /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     5933..5952
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6171..6275)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6350..6369)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6541..6920
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6922..7120
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             7138..7318
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7365..7490
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7987..8220
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8405..8449
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
ORIGIN
        1 aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc
       61 ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa
      121 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
      181 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
      241 gggacagcag agatccagtt tggttagtac cgggcccgct ctagacgtat taccgccatg
      301 cattagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
      361 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      421 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      481 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      541 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      601 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      661 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
      721 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
      781 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
      841 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg
      901 ctaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      961 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     1021 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1081 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     1141 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1201 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     1261 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1321 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     1381 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     1441 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1501 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     1561 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1621 gagctgtaca agtagcggcc gcaagccttg ttaagtgctc gcttcggcag cacatatact
     1681 atgttgaatg aggcttcagt actttacaga atcgttgcct gcacatcttg gaaacacttg
     1741 ctgggattac ttcttcaggt taacccaaca gaaggctcga gaaggtatat tgctgttgac
     1801 agtgagcgca cactgagcct cgttctccat tagtgaagcc acagatgtaa tggagaacga
     1861 ggctcagtgt atgcctactg cctcggaatt caaggggcta ctttaggagc aattatcttg
     1921 tttactaaaa ctgaatacct tgctatctct ttgatacatt tttacaaagc tgaattaaaa
     1981 tggtataaat taaatcactt ttttcaattg gaagactaat gcgtttaaac acgcggcgac
     2041 gcgttcgacc gaataaatac ctgtgacgga agatcacttc gcagaataaa taaatcctgg
     2101 tgtccctgtt gataccggga agccctgggc caacttttgg cgaaaatgag acgttgatcg
     2161 gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt
     2221 tgagttgtcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
     2281 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
     2341 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
     2401 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
     2461 tgctcatccg gaattacgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
     2521 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
     2581 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
     2641 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
     2701 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
     2761 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
     2821 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
     2881 acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca gttattggtg
     2941 cccttaaacg cctggttgct acgcctgaat aagtgataat aagcggatga atggcagaaa
     3001 ttcggatctc gcgcgtttgg gcggtggctc cctgccacgc ggctccgaac agaagctgat
     3061 ctccgaagag gatctgacat gtgtttaaac ttaattaagt cgaggtcgac ggtatcgata
     3121 agctcgcttc acgagatcat gtttaagggt tccggttcca ctaggtacaa ttcgatatca
     3181 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     3241 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     3301 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     3361 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     3421 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     3481 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     3541 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     3601 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     3661 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     3721 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     3781 atcgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac aagtagcaat
     3841 acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt
     3901 tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt
     3961 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     4021 gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg gcagaactac
     4081 acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa gctagtacca
     4141 gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt gttacaccct
     4201 gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag gtttgacagc
     4261 cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg gtctctctgg
     4321 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     4381 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     4441 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag catgtgagca
     4501 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4561 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4621 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4681 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4741 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4801 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4861 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4921 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4981 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5041 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5101 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5161 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5221 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5281 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5341 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5401 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5461 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5521 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5581 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5641 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5701 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5761 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5821 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5881 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5941 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6001 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6061 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6121 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6181 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6241 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6301 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     6361 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     6421 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     6481 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
     6541 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     6601 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     6661 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     6721 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     6781 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     6841 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     6901 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     6961 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     7021 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     7081 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
     7141 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     7201 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     7261 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     7321 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
     7381 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
     7441 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
     7501 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     7561 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     7621 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     7681 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     7741 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     7801 acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg atcttcagac ctggaggagg
     7861 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     7921 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     7981 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     8041 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     8101 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     8161 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     8221 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     8281 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     8341 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     8401 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     8461 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     8521 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     8581 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaagg
//
