LOCUS       Exported                8637 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Encodes short hairpin RNA (shRNA) #2 that targets the 
            3'-untranslated region of the rat Pdyn gene.
ACCESSION   .
VERSION     .
KEYWORDS    pPRIME-CMV-GFP-shDyn2
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 13, 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"
     regulatory      10..19
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             16..732
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(61..82)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(322..341)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     669..690
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           784..905
                     /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           933..947
                     /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           975..1101
                     /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        1255..1357
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1358..2017
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1424..1443)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2147..2176
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     2201..2217
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    2292..2880
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2345..2365)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2763..2774)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2883..2899)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             3409..3589
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(3651..4239)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3731..3750)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4410..5270)
                     /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     5033..5052
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5271..5375)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5450..5469)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5641..6020
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6022..6220
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             6238..6418
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6465..6590
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7087..7320
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7505..7549
                     /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"
     misc_feature    7847..7964
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        8101..8404
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8405..8608
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 ctaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
       61 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
      121 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
      181 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
      241 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
      301 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
      361 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
      421 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
      481 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
      541 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
      601 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
      661 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
      721 gagctgtaca agtagcggcc gcaagccttg ttaagtgctc gcttcggcag cacatatact
      781 atgttgaatg aggcttcagt actttacaga atcgttgcct gcacatcttg gaaacacttg
      841 ctgggattac ttcttcaggt taacccaaca gaaggctcga gaaggtatat tgctgttgac
      901 agtgagcgcg ctcttcatgt gttctgaaat tagtgaagcc acagatgtaa tttcagaaca
      961 catgaagagc ttgcctactg cctcggaatt caaggggcta ctttaggagc aattatcttg
     1021 tttactaaaa ctgaatacct tgctatctct ttgatacatt tttacaaagc tgaattaaaa
     1081 tggtataaat taaatcactt ttttcaattg gaagactaat gcgtttaaac acgcggcgac
     1141 gcgttcgacc gaataaatac ctgtgacgga agatcacttc gcagaataaa taaatcctgg
     1201 tgtccctgtt gataccggga agccctgggc caacttttgg cgaaaatgag acgttgatcg
     1261 gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt
     1321 tgagttgtcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
     1381 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
     1441 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
     1501 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
     1561 tgctcatccg gaattacgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
     1621 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
     1681 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
     1741 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
     1801 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
     1861 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
     1921 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
     1981 acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca gttattggtg
     2041 cccttaaacg cctggttgct acgcctgaat aagtgataat aagcggatga atggcagaaa
     2101 ttcggatctc gcgcgtttgg gcggtggctc cctgccacgc ggctccgaac agaagctgat
     2161 ctccgaagag gatctgacat gtgtttaaac ttaattaagt cgaggtcgac ggtatcgata
     2221 agctcgcttc acgagatcat gtttaagggt tccggttcca ctaggtacaa ttcgatatca
     2281 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     2341 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     2401 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     2461 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     2521 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     2581 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     2641 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     2701 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     2761 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     2821 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     2881 atcgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac aagtagcaat
     2941 acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt
     3001 tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt
     3061 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     3121 gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg gcagaactac
     3181 acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa gctagtacca
     3241 gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt gttacaccct
     3301 gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag gtttgacagc
     3361 cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg gtctctctgg
     3421 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     3481 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     3541 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag catgtgagca
     3601 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3661 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3721 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3781 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3841 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3901 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3961 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4021 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4081 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4141 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4201 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4261 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4321 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4381 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4441 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4501 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4561 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4621 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4681 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4741 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4801 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4861 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4921 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4981 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5041 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5101 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5161 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5221 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5281 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5341 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5401 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     5461 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     5521 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     5581 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
     5641 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     5701 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     5761 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     5821 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     5881 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     5941 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     6001 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     6061 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     6121 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     6181 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
     6241 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     6301 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     6361 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     6421 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
     6481 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
     6541 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
     6601 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     6661 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     6721 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     6781 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     6841 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     6901 acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg atcttcagac ctggaggagg
     6961 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     7021 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     7081 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     7141 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     7201 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     7261 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     7321 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     7381 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     7441 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     7501 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     7561 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     7621 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     7681 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
     7741 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
     7801 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     7861 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     7921 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     7981 acagcagaga tccagtttgg ttagtaccgg gcccgctcta gacgtattac cgccatgcat
     8041 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     8101 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     8161 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     8221 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     8281 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     8341 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     8401 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     8461 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     8521 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     8581 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcg
//
