LOCUS       sequence-254920-        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.PAPD4-3
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1447..1680
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1865..1909
                     /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    2207..2324
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2577..2610)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2614..2634
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        2879..3090
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          3091..4029
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3986..4006
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4050..4646
                     /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(4050..4069)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4546..4566
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4656..4709
                     /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             4722..5420
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(5440..5473)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5529..6117
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5582..5602)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6000..6011)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6120..6136)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6121..6137)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6646..6826
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6888..7476)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6968..6987)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7647..8438,8439..8507))
                     /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     8270..8289
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8508..8612)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8687..8706)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg atcttcagac ctggaggagg
     1321 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     1381 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     1441 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     1501 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     1561 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     1621 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     1681 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     1741 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     1801 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     1861 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     1921 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     1981 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     2041 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
     2101 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
     2161 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     2221 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2281 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2341 acagcagaga tccagtttgg ttagtaccgg gcccgctcta gagatccgac gcgccatctc
     2401 taggcccgcg ccggccccct cgcacggact tgtgggagaa gctcggctac tcccctgccc
     2461 cggttaattt gcatataata tttcctagta actatagagg cttaatgtgc gataaaagac
     2521 agataatctg ttctttttaa tactagctac attttacatg ataggcttgg atttctataa
     2581 cttcgtatag catacattat acgaagttat aaacagcaca aaaggaaact caccctaact
     2641 gtaaagtaat tgtgtgtttt gagactataa gtatcccttg gagaaccacc ttgttggccc
     2701 ggatgagaat aggcaggttt aagagctaag ctggaaacag catagcaagt ttaaataagg
     2761 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc tcgagtacta
     2821 ggatccatta ggcggccgcg tggataaccg tattaccgcc atgcatgtgc ccgtcagtgg
     2881 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc
     2941 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
     3001 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
     3061 tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct
     3121 ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac
     3181 gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct
     3241 taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc
     3301 gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
     3361 taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg
     3421 ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt
     3481 gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga
     3541 cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc
     3601 gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg
     3661 ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
     3721 gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt
     3781 gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgcg cttttggagt
     3841 acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg
     3901 gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt
     3961 tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt
     4021 ccatttcagg tgtcgtgagc tagcccacca tgaccgagta caagcccacg gtgcgcctcg
     4081 ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc cgccgcgttc gccgactacc
     4141 ccgccacgcg ccacaccgtc gacccggacc gccacatcga gcgggtcacc gagctgcaag
     4201 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     4261 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     4321 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     4381 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
     4441 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     4501 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     4561 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     4621 ggtgcatgac ccgcaagccc ggtgccggat cgggagaggg cagaggaagt ctgctaacat
     4681 gcggtgacgt cgaggagaat cctggcccac cggtcgccac cagcgagctg attaaggaga
     4741 acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc aagtgcacat
     4801 ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag gtggtcgagg
     4861 gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac ggcagcaaga
     4921 ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc cctgagggct
     4981 tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca
     5041 ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcacat
     5101 ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc gagacgctgt
     5161 accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc gtgggcggga
     5221 gccatctgat cgcaaacatc aagaccacat atagatccaa gaaacccgct aagaacctca
     5281 agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag gccaacaacg
     5341 agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc cctagcaaac
     5401 tggggcacaa gcttaattaa gaattcgtcg agggacctaa taacttcgta tagcatacat
     5461 tatacgaagt tatacatgtt taagggttcc ggttccacta ggtacaattc gatatcaagc
     5521 ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     5581 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     5641 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     5701 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     5761 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     5821 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     5881 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     5941 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     6001 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     6061 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     6121 gataccgtcg acctcgatcg agacctagaa aaacatggag caatcacaag tagcaataca
     6181 gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt
     6241 ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
     6301 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     6361 atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca
     6421 ccagggccag ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt
     6481 gagcaagaga aggtagaaga agccaatgaa ggagagaaca cccgcttgtt acaccctgtg
     6541 agcctgcatg ggatggatga cccggagaga gaagtattag agtggaggtt tgacagccgc
     6601 ctagcatttc atcacatggc ccgagagctg catccggact gtactgggtc tctctggtta
     6661 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     6721 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     6781 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa
     6841 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6901 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6961 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     7021 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     7081 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     7141 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     7201 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     7261 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     7321 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     7381 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     7441 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     7501 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     7561 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     7621 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     7681 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     7741 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     7801 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7861 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7921 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7981 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     8041 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     8101 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     8161 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     8221 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     8281 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     8341 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     8401 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     8461 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     8521 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     8581 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     8641 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     8701 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     8761 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     8821 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgtt
//
