LOCUS       Exported                8637 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Encodes short hairpin RNA (shRNA) #1 that targets the the 
            3'-untranslated region of the rat Nts gene.
ACCESSION   .
VERSION     .
KEYWORDS    pPRIME-CMV-GFP-shNts1
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"
     misc_feature    189..306
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        443..746
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        747..950
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      989..998
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             995..1711
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1040..1061)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1301..1320)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1648..1669
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           1763..1884
                     /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           1912..1926
                     /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           1954..2080
                     /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        2234..2336
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             2337..2996
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2403..2422)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3126..3155
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     3180..3196
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3271..3859
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3324..3344)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3742..3753)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3862..3878)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             4388..4568
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4630..5218)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4710..4729)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5389..6249)
                     /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     6012..6031
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6250..6354)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6429..6448)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6620..6999
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7001..7199
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             7217..7397
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7444..7569
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8066..8299
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8484..8528
                     /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 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
       61 gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca
      121 ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc
      181 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
      241 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
      301 aattttcggg tttattacag ggacagcaga gatccagttt ggttagtacc gggcccgctc
      361 tagacgtatt accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc
      421 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      541 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      601 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      661 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      721 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      781 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      841 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      901 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga
      961 accgtcagat ccgctagcgc taccggtcgc caccatggtg agcaagggcg aggagctgtt
     1021 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     1081 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     1141 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     1201 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     1261 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     1321 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1381 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     1441 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     1501 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     1561 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     1621 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     1681 gatcactctc ggcatggacg agctgtacaa gtagcggccg caagccttgt taagtgctcg
     1741 cttcggcagc acatatacta tgttgaatga ggcttcagta ctttacagaa tcgttgcctg
     1801 cacatcttgg aaacacttgc tgggattact tcttcaggtt aacccaacag aaggctcgag
     1861 aaggtatatt gctgttgaca gtgagcgcca tgtgattctc atcctttact agtgaagcca
     1921 cagatgtagt aaaggatgag aatcacatgt tgcctactgc ctcggaattc aaggggctac
     1981 tttaggagca attatcttgt ttactaaaac tgaatacctt gctatctctt tgatacattt
     2041 ttacaaagct gaattaaaat ggtataaatt aaatcacttt tttcaattgg aagactaatg
     2101 cgtttaaaca cgcggcgacg cgttcgaccg aataaatacc tgtgacggaa gatcacttcg
     2161 cagaataaat aaatcctggt gtccctgttg ataccgggaa gccctgggcc aacttttggc
     2221 gaaaatgaga cgttgatcgg cacgtaagag gttccaactt tcaccataat gaaataagat
     2281 cactaccggg cgtatttttt gagttgtcga gattttcagg agctaaggaa gctaaaatgg
     2341 agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt
     2401 ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta
     2461 cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca
     2521 ttcttgcccg cctgatgaat gctcatccgg aattacgtat ggcaatgaaa gacggtgagc
     2581 tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt
     2641 tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc
     2701 aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata
     2761 tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca
     2821 atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca
     2881 aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg
     2941 gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg gcgtaatttt
     3001 tttaaggcag ttattggtgc ccttaaacgc ctggttgcta cgcctgaata agtgataata
     3061 agcggatgaa tggcagaaat tcggatctcg cgcgtttggg cggtggctcc ctgccacgcg
     3121 gctccgaaca gaagctgatc tccgaagagg atctgacatg tgtttaaact taattaagtc
     3181 gaggtcgacg gtatcgataa gctcgcttca cgagatcatg tttaagggtt ccggttccac
     3241 taggtacaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     3301 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     3361 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3421 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3481 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3541 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     3601 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     3661 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     3721 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     3781 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     3841 ctttgggccg cctccccgca tcgataccgt cgacctcgat cgagacctag aaaaacatgg
     3901 agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc tagaagcaca
     3961 agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac caatgactta
     4021 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     4081 tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac aaggctactt
     4141 ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga cctttggatg
     4201 gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa
     4261 cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtatt
     4321 agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc tgcatccgga
     4381 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     4441 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     4501 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     4561 ctctagcagc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4621 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4681 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4741 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4801 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4861 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4921 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4981 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5041 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5101 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5161 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5221 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5281 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5341 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5401 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5461 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5521 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5581 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5641 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5701 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5761 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5821 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5881 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5941 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6001 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6061 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6121 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6181 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6241 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6301 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6361 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
     6421 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
     6481 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     6541 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     6601 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
     6661 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     6721 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     6781 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     6841 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     6901 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     6961 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     7021 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     7081 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     7141 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcagc
     7201 gcgttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     7261 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     7321 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     7381 gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag
     7441 gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg
     7501 cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc
     7561 gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg
     7621 ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa
     7681 cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga
     7741 cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta
     7801 gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac
     7861 aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cggccgctga
     7921 tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata
     7981 aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc
     8041 agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag
     8101 gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg
     8161 gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc
     8221 aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc
     8281 taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg
     8341 ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga
     8401 cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg
     8461 aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg
     8521 caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa
     8581 tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtga
//
