LOCUS       sequence-132437-        8567 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dsRed and shRNA against vgat under the control of the CMV 
            promoter in eukaryotic cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8567)
  AUTHORS   Yu X, Ye Z, Houston CM, Zecharia AY, Ma Y, Zhang Z, Uygun DS, Parker
            S, Vyssotski AL, Yustos R, Franks NP, Brickley SG, Wisden W
  TITLE     Wakefulness Is Governed by GABA and Histamine Cotransmission.
  JOURNAL   Neuron. 2015 Jun 17. pii: S0896-6273(15)00516-4. doi: 
            10.1016/j.neuron.2015.06.003.
  PUBMED    26094607
REFERENCE   2  (bases 1 to 8567)
  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: "Neuron. 
            2015 Jun 17. pii: S0896-6273(15)00516-4. doi: 
            10.1016/j.neuron.2015.06.003."
COMMENT     Sequence Label: pPRIME-dsRed-shvgat
FEATURES             Location/Qualifiers
     source          1..8567
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    1023..1148
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1645..1878
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2063..2107
                     /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    2405..2522
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2659..2962
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2963..3166
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3212..3889
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MDSSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(3342..3360)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(3392..3412)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3610..3629
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3804..3827
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     ncRNA           3939..4060
                     /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           4088..4102
                     /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           4130..4256
                     /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        4410..4512
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4513..5172
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4579..4598)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             5304..5333
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    5456..6044
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5509..5529)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5927..5938)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6047..6063)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             6573..6753
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6815..7403)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6895..6914)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7574..8365,8366..8434))
                     /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     8197..8216
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8435..8539)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata gaccagatct gagcctggga gctctctggc
      841 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
      901 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
      961 tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg
     1021 agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc
     1081 gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg
     1141 agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc
     1201 cagggggaaa gaaaaaatat aaataaacat atagtatggg caagcaggga gctagaacga
     1261 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
     1321 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
     1381 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     1441 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgcgctgat
     1501 cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa
     1561 agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca
     1621 gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg
     1681 aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg
     1741 tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca
     1801 actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct
     1861 aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc
     1921 tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac
     1981 ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga
     2041 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
     2101 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
     2161 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
     2221 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc
     2281 cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg
     2341 attagtgaac ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc
     2401 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
     2461 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
     2521 ttcgggttta ttacagggac agcagagatc cagtttggtt agtaccgggc ccgctctaga
     2581 cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag
     2641 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     2701 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     2761 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     2821 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     2881 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     2941 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     3001 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     3061 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     3121 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
     3181 tcagatccgc tagcgctacc gggcctgcag gatggactcc tccgaggacg tcatcaagga
     3241 gttcatgcgc ttcaaggtgc gcatggaggg ctccgtgaac ggccacgagt tcgagatcga
     3301 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     3361 gggcggcccc ctgcccttcg cctgggacat cctgtccccc cagttccagt acggctccaa
     3421 ggtgtacgtg aagcaccccg ccgacatccc cgactacaag aagctgtcct tccccgaggg
     3481 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
     3541 ctcctccctg caggacggct ccttcatcta caaggtgaag ttcatcggcg tgaacttccc
     3601 ctccgacggc cccgtaatgc agaagaagac tatgggctgg gaggcctcca ccgagcgcct
     3661 gtacccccgc gacggcgtgc tgaagggcga gatccacaag gccctgaagc tgaaggacgg
     3721 cggccactac ctggtggagt tcaagtccat ctacatggcc aagaagcccg tgcagctgcc
     3781 cggctactac tacgtggact ccaagctgga catcacctcc cacaacgagg actacaccat
     3841 cgtggagcag tacgagcgcg ccgagggccg ccaccacctg ttcctgtagg cggccgcaag
     3901 ccttgttaag tgctcgcttc ggcagcacat atactatgtt gaatgaggct tcagtacttt
     3961 acagaatcgt tgcctgcaca tcttggaaac acttgctggg attacttctt caggttaacc
     4021 caacagaagg ctcgagaagg tatattgctg ttgacagtga gcgcactcat cttgtgcaat
     4081 gtatctagtg aagccacaga tgtagataca ttgcacaaga tgagtttgcc tactgcctcg
     4141 gaattcaagg ggctacttta ggagcaatta tcttgtttac taaaactgaa taccttgcta
     4201 tctctttgat acatttttac aaagctgaat taaaatggta taaattaaat cacttttttc
     4261 aattggaaga ctaatgcgtt taaacacgcg gcgacgcgtt cgaccgaata aatacctgtg
     4321 acggaagatc acttcgcaga ataaataaat cctggtgtcc ctgttgatac cgggaagccc
     4381 tgggccaact tttggcgaaa atgagacgtt gatcggcacg taagaggttc caactttcac
     4441 cataatgaaa taagatcact accgggcgta ttttttgagt tgtcgagatt ttcaggagct
     4501 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
     4561 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
     4621 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
     4681 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt acgtatggca
     4741 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat
     4801 gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt
     4861 ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa
     4921 gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt caccagtttt
     4981 gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat gggcaaatat
     5041 tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt
     5101 gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag
     5161 ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg ttgctacgcc
     5221 tgaataagtg ataataagcg gatgaatggc agaaattcgg atctcgaccg cgtttgggcg
     5281 gtggctccct gccacgcggc tccgaacaga agctgatctc cgaagaggat ctgacatgtg
     5341 tttaaacctc gacttaatta agtcgagggt cgacggtatc gataagctcg cttcacgaga
     5401 tcatgtttaa gggttccggt tccactaggt acaattcgat atcaagctta tcgataatca
     5461 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     5521 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     5581 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     5641 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     5701 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     5761 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     5821 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
     5881 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     5941 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     6001 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc
     6061 tcgatcgaga cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg
     6121 ctgattgtgc ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc
     6181 aggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     6241 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt
     6301 ggatctacca cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga
     6361 tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg
     6421 tagaagaagc caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga
     6481 tggatgaccc ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc
     6541 acatggcccg agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag
     6601 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     6661 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     6721 gaccctttta gtcagtgtgg aaaatctcta gcagcatgtg agcaaaaggc cagcaaaagg
     6781 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6841 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6901 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6961 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     7021 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     7081 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     7141 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     7201 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     7261 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     7321 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     7381 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     7441 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     7501 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     7561 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     7621 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     7681 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     7741 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     7801 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     7861 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7921 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7981 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     8041 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     8101 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     8161 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     8221 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     8281 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     8341 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     8401 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     8461 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     8521 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgac
//
