LOCUS       sequence-254146-        8877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PAPD4 gRNA for CRISPRi.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8877)
  AUTHORS   Kleaveland B, Shi CY, Stefano J, Bartel DP
  TITLE     A Network of Noncoding Regulatory RNAs Acts in the Mammalian Brain.
  JOURNAL   Cell. 2018 Jul 12;174(2):350-362.e17. doi: 
            10.1016/j.cell.2018.05.022. Epub 2018 Jun 7.
  PUBMED    29887379
REFERENCE   2  (bases 1 to 8877)
  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: 
            "10.1016/j.cell.2018.05.022"; journalName: "Cell"; date: 
            "2018-07-12- 12"; volume: "174"; issue: "2"; pages: "350-362.e17"
COMMENT     Sequence Label: pU6-sgRNA EF1Alpha-puro-T2A-BFP.PAPD4-2
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..200
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             218..398
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    445..570
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1067..1300
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1485..1529
                     /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    1827..1944
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2197..2230)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2234..2254
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        2499..2710
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          2711..3649
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3606..3626
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3670..4266
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3670..3689)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4166..4186
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4276..4329
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4342..5040
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(5060..5093)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5149..5737
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5202..5222)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5620..5631)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5740..5756)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5741..5757)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6266..6446
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6508..7096)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6588..6607)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7267..8058,8059..8127))
                     /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     7890..7909
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8128..8232)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8307..8326)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        8498..8877
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag cgcgttttgc ctgtactggg tctctctggt tagaccagat
      241 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
      301 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
      361 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg
      421 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc
      481 acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc
      541 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga
      601 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta
      661 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa
      721 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt
      781 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa
      841 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca
      901 cagcaagcgg ccggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
      961 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     1021 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     1081 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     1141 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     1201 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     1261 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     1321 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     1381 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     1441 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     1501 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     1561 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     1621 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     1681 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     1741 gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg ccaattctgc
     1801 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     1861 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     1921 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg
     1981 ttagtaccgg gcccgctcta gagatccgac gcgccatctc taggcccgcg ccggccccct
     2041 cgcacggact tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata
     2101 tttcctagta actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa
     2161 tactagctac attttacatg ataggcttgg atttctataa cttcgtatag catacattat
     2221 acgaagttat aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt
     2281 gagactataa gtatcccttg gagaaccacc ttgttgggga cagctaggcc cagctcgttt
     2341 aagagctaag ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga
     2401 aaaagtggca ccgagtcggt gctttttttc tcgagtacta ggatccatta ggcggccgcg
     2461 tggataaccg tattaccgcc atgcatgtgc ccgtcagtgg gcagagcgca catcgcccac
     2521 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
     2581 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
     2641 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
     2701 cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg
     2761 cccttgcgtg ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct
     2821 tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg
     2881 tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct
     2941 tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc
     3001 tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg
     3061 tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc
     3121 ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg
     3181 ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag
     3241 gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc
     3301 agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca
     3361 aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc
     3421 gccgtccagg cacctcgatt agttctcgcg cttttggagt acgtcgtctt taggttgggg
     3481 ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc
     3541 agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt
     3601 cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgagc
     3661 tagcccacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     3721 cgggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     3781 gacccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     3841 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     3901 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     3961 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     4021 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     4081 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     4141 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     4201 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     4261 ggtgccggat cgggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat
     4321 cctggcccac cggtcgccac cagcgagctg attaaggaga acatgcacat gaagctgtac
     4381 atggagggca ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc
     4441 tacgagggca cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc
     4501 ttcgacatcc tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag
     4561 ggcatccccg acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc
     4621 acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc
     4681 ctcatctaca acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag
     4741 aagaaaacac tcggctggga ggccttcacc gagacgctgt accccgctga cggcggcctg
     4801 gaaggcagaa acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacatc
     4861 aagaccacat atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat
     4921 gtggactaca gactggaaag aatcaaggag gccaacaacg agacctacgt cgagcagcac
     4981 gaggtggcag tggccagata ctgcgacctc cctagcaaac tggggcacaa gcttaattaa
     5041 gaattcgtcg agggacctaa taacttcgta tagcatacat tatacgaagt tatacatgtt
     5101 taagggttcc ggttccacta ggtacaattc gatatcaagc ttatcgataa tcaacctctg
     5161 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     5221 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     5281 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     5341 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     5401 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     5461 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     5521 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     5581 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     5641 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     5701 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg acctcgatcg
     5761 agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca atgctgattg
     5821 tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac ctcaggtacc
     5881 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     5941 gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc tgtggatcta
     6001 ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag ggatcagata
     6061 tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga aggtagaaga
     6121 agccaatgaa ggagagaaca cccgcttgtt acaccctgtg agcctgcatg ggatggatga
     6181 cccggagaga gaagtattag agtggaggtt tgacagccgc ctagcatttc atcacatggc
     6241 ccgagagctg catccggact gtactgggtc tctctggtta gaccagatct gagcctggga
     6301 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     6361 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     6421 ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa aggccaggaa
     6481 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     6541 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     6601 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6661 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6721 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6781 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6841 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6901 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6961 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     7021 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     7081 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     7141 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     7201 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     7261 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     7321 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     7381 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     7441 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     7501 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     7561 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     7621 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7681 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7741 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7801 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7861 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7921 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7981 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     8041 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     8101 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     8161 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     8221 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
     8281 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     8341 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
     8401 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
     8461 gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat
     8521 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     8581 taacttacgg taaatggccc gcctagctga ccgcccaacg acccccgccc attgacgtca
     8641 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     8701 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     8761 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     8821 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatg
//
