LOCUS       sequence-189625-        7687 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a shRNA targeting the rat P2rx4 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7687)
  AUTHORS   Franklin KM, Hauser SR, Lasek AW, Bell RL, McBride WJ
  TITLE     Involvement of Purinergic P2X4 Receptors in Alcohol Intake of 
            High-Alcohol-Drinking (HAD) Rats.
  JOURNAL   Alcohol Clin Exp Res. 2015 Oct;39(10):2022-31. doi: 
            10.1111/acer.12836. Epub 2015 Sep 3.
  PUBMED    26334550
REFERENCE   2  (bases 1 to 7687)
  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"; doi: 
            "10.1111/acer.12836"; journalName: "Alcohol Clin Exp Res"; date: 
            "2015-10"; volume: "39"; issue: "10"; pages: "2022-31"
COMMENT     Sequence Label: pLV-shP2rx4-1026
FEATURES             Location/Qualifiers
     source          1..7687
                     /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"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /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    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2620..2932
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2851..2871
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     2989..3005
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3047..3080)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        3196..3499
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3500..3703
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      3742..3751
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3748..3750,3751..3753,3754..4467)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3748..3750,3751..3753,3754..4464)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3793..3814)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4054..4073)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4401..4422
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4487..4520)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4576..5164
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4629..4649)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5047..5058)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5167..5183)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             5693..5873
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5935..6523)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6015..6034)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6694..7485,7486..7554))
                     /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     7317..7336
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7555..7659)
                     /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 taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta gtaccgggcc cgctctagag atccgacgcg ccatctctag
     2641 gcccgcgccg gccccctcgc acagacttgt gggagaagct cggctactcc cctgccccgg
     2701 ttaatttgca tataatattt cctagtaact atagaggctt aatgtgcgat aaaagacaga
     2761 taatctgttc tttttaatac tagctacatt ttacatgata ggcttggatt tctataagag
     2821 atacaaatac taaattatta ttttaaaaaa cagcacaaaa ggaaactcac cctaactgta
     2881 aagtaattgt gtgttttgag actataaata tcccttggag aaaagccttg tttgcacact
     2941 caccaaggcg tattcaagag atacgccttg gtgagtgtgc ttttttctcg aggtcgacgg
     3001 tatcgataag ctcgcttcac gagattccag caggtcgagg gacctaataa cttcgtatag
     3061 catacattat acgaagttat attaagggtt ccaagcttaa gcggccgcgt ggataaccgt
     3121 attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag ttcatagccc
     3181 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     3241 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     3301 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     3361 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     3421 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     3481 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     3541 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     3601 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     3661 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
     3721 gatccgctag cgctaccggt cgccaccatg gtgagcaagg gcgaggagct gttcaccggg
     3781 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     3841 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     3901 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     3961 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     4021 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     4081 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     4141 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     4201 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     4261 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     4321 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     4381 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     4441 ctcggcatgg acgagctgta caagtaggaa ttcgtcgagg gacctaataa cttcgtatag
     4501 catacattat acgaagttat acatgtttaa gggttccggt tccactaggt acaattcgat
     4561 atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     4621 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     4681 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     4741 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     4801 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     4861 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     4921 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     4981 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     5041 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     5101 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     5161 ccgcatcgat accgtcgacc tcgatcgaga cctagaaaaa catggagcaa tcacaagtag
     5221 caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg aggaggaggt
     5281 gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg cagctgtaga
     5341 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     5401 acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg attggcagaa
     5461 ctacacacca gggccaggga tcagatatcc actgaccttt ggatggtgct acaagctagt
     5521 accagttgag caagagaagg tagaagaagc caatgaagga gagaacaccc gcttgttaca
     5581 ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtattagagt ggaggtttga
     5641 cagccgccta gcatttcatc acatggcccg agagctgcat ccggactgta ctgggtctct
     5701 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     5761 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     5821 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagcatgtg
     5881 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5941 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     6001 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     6061 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6121 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6181 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6241 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6301 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6361 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6421 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6481 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6541 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6601 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6661 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6721 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6781 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6841 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6901 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6961 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     7021 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7081 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7141 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7201 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7261 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7321 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7381 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7441 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7501 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7561 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     7621 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     7681 acctgac
//
