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 Nts.
ACCESSION   .
VERSION     .
KEYWORDS    pPRIME-CMV-GFP-shNts2
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"
     primer_bind     151..172
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           266..387
                     /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           415..429
                     /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           457..583
                     /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        737..839
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             840..1499
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(906..925)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1629..1658
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     1683..1699
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1774..2362
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1827..1847)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2245..2256)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2365..2381)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             2891..3071
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(3133..3721)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3213..3232)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3892..4752)
                     /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     4515..4534
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4753..4857)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4932..4951)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5123..5502
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5504..5702
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             5720..5900
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5947..6072
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6569..6802
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6987..7031
                     /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    7329..7446
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        7583..7886
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7887..8090
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      8129..8138
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             8135..214
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(8180..8201)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(8441..8460)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
       61 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
      121 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
      181 cgggatcact ctcggcatgg acgagctgta caagtagcgg ccgcaagcct tgttaagtgc
      241 tcgcttcggc agcacatata ctatgttgaa tgaggcttca gtactttaca gaatcgttgc
      301 ctgcacatct tggaaacact tgctgggatt acttcttcag gttaacccaa cagaaggctc
      361 gagaaggtat attgctgttg acagtgagcg cacctgttat ctggatacac attagtgaag
      421 ccacagatgt aatgtgtatc cagataacag gtatgcctac tgcctcggaa ttcaaggggc
      481 tactttagga gcaattatct tgtttactaa aactgaatac cttgctatct ctttgataca
      541 tttttacaaa gctgaattaa aatggtataa attaaatcac ttttttcaat tggaagacta
      601 atgcgtttaa acacgcggcg acgcgttcga ccgaataaat acctgtgacg gaagatcact
      661 tcgcagaata aataaatcct ggtgtccctg ttgataccgg gaagccctgg gccaactttt
      721 ggcgaaaatg agacgttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa
      781 gatcactacc gggcgtattt tttgagttgt cgagattttc aggagctaag gaagctaaaa
      841 tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac
      901 attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata
      961 ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc
     1021 acattcttgc ccgcctgatg aatgctcatc cggaattacg tatggcaatg aaagacggtg
     1081 agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa
     1141 cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt
     1201 cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga
     1261 atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg
     1321 ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg
     1381 acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg
     1441 tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaat
     1501 ttttttaagg cagttattgg tgcccttaaa cgcctggttg ctacgcctga ataagtgata
     1561 ataagcggat gaatggcaga aattcggatc tcgcgcgttt gggcggtggc tccctgccac
     1621 gcggctccga acagaagctg atctccgaag aggatctgac atgtgtttaa acttaattaa
     1681 gtcgaggtcg acggtatcga taagctcgct tcacgagatc atgtttaagg gttccggttc
     1741 cactaggtac aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt
     1801 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     1861 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     1921 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     1981 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     2041 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     2101 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     2161 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
     2221 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     2281 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     2341 tccctttggg ccgcctcccc gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca
     2401 tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc
     2461 acaagaggag gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac
     2521 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     2581 aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta
     2641 cttccctgat tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg
     2701 atggtgctac aagctagtac cagttgagca agagaaggta gaagaagcca atgaaggaga
     2761 gaacacccgc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt
     2821 attagagtgg aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc
     2881 ggactgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     2941 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     3001 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     3061 aatctctagc agcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3121 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3181 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3241 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3301 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3361 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3421 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3481 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3541 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     3601 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     3661 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     3721 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     3781 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3841 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     3901 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3961 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     4021 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     4081 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4141 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4201 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4261 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     4321 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     4381 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     4441 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     4501 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4561 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     4621 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     4681 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     4741 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4801 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     4861 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     4921 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     4981 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
     5041 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
     5101 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     5161 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     5221 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     5281 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     5341 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     5401 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     5461 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     5521 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     5581 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     5641 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     5701 agcgcgtttt gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     5761 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     5821 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     5881 agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca
     5941 gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg
     6001 cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg
     6061 tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta
     6121 aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta
     6181 gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg
     6241 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
     6301 gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta
     6361 gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccggccgc
     6421 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
     6481 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
     6541 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
     6601 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
     6661 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
     6721 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
     6781 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
     6841 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
     6901 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
     6961 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat
     7021 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
     7081 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga
     7141 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
     7201 gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca
     7261 ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc
     7321 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     7381 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     7441 aattttcggg tttattacag ggacagcaga gatccagttt ggttagtacc gggcccgctc
     7501 tagacgtatt accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc
     7561 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     7621 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     7681 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     7741 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     7801 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     7861 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     7921 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     7981 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     8041 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga
     8101 accgtcagat ccgctagcgc taccggtcgc caccatggtg agcaagggcg aggagctgtt
     8161 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     8221 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     8281 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     8341 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     8401 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     8461 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     8521 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     8581 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagat
//
