LOCUS       sequence-273592-        8877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CENPB CRISPRi guide RNA 6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8877)
  AUTHORS   Wu X, Bartel DP
  TITLE     Widespread Influence of 3'-End Structures on Mammalian mRNA 
            Processing and Stability.
  JOURNAL   Cell. 2017 May 18;169(5):905-917.e11. doi: 
            10.1016/j.cell.2017.04.036.
  PUBMED    28525757
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.2017.04"; journalName: "Cell"; date: "2017-05-18- 
            18"; volume: "169"; issue: "5"; pages: "905-917.e11"
COMMENT     Sequence Label: CRISPRi-CENPB-g6
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    167..284
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(537..570)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     574..594
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        839..1050
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1051..1989
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1946..1966
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2010..2606
                     /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(2010..2029)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2506..2526
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2616..2669
                     /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             2682..3380
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(3400..3433)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3489..4077
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3542..3562)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3960..3971)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4080..4096)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4081..4097)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4606..4786
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4848..5436)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4928..4947)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5607..6398,6399..6467))
                     /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     6230..6249
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6468..6572)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6647..6666)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6838..7217
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7219..7417
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7372..7392
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             7435..7615
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7662..7787
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8284..8517
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8702..8746
                     /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"
ORIGIN
        1 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
       61 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
      121 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
      181 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
      241 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
      301 acagcagaga tccagtttgg ttagtaccgg gcccgctcta gagatccgac gcgccatctc
      361 taggcccgcg ccggccccct cgcacggact tgtgggagaa gctcggctac tcccctgccc
      421 cggttaattt gcatataata tttcctagta actatagagg cttaatgtgc gataaaagac
      481 agataatctg ttctttttaa tactagctac attttacatg ataggcttgg atttctataa
      541 cttcgtatag catacattat acgaagttat aaacagcaca aaaggaaact caccctaact
      601 gtaaagtaat tgtgtgtttt gagactataa gtatcccttg gagaaccacc ttgttggcgg
      661 aaggcaggaa gtgccggttt aagagctaag ctggaaacag catagcaagt ttaaataagg
      721 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc tcgagtacta
      781 ggatccatta ggcggccgcg tggataaccg tattaccgcc atgcatgtgc ccgtcagtgg
      841 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc
      901 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
      961 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
     1021 tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct
     1081 ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac
     1141 gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct
     1201 taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc
     1261 gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
     1321 taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg
     1381 ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt
     1441 gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga
     1501 cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc
     1561 gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg
     1621 ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
     1681 gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt
     1741 gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgcg cttttggagt
     1801 acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg
     1861 gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt
     1921 tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt
     1981 ccatttcagg tgtcgtgagc tagcccacca tgaccgagta caagcccacg gtgcgcctcg
     2041 ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc cgccgcgttc gccgactacc
     2101 ccgccacgcg ccacaccgtc gacccggacc gccacatcga gcgggtcacc gagctgcaag
     2161 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     2221 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     2281 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     2341 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
     2401 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     2461 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     2521 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     2581 ggtgcatgac ccgcaagccc ggtgccggat cgggagaggg cagaggaagt ctgctaacat
     2641 gcggtgacgt cgaggagaat cctggcccac cggtcgccac cagcgagctg attaaggaga
     2701 acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc aagtgcacat
     2761 ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag gtggtcgagg
     2821 gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac ggcagcaaga
     2881 ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc cctgagggct
     2941 tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca
     3001 ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcacat
     3061 ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc gagacgctgt
     3121 accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc gtgggcggga
     3181 gccatctgat cgcaaacatc aagaccacat atagatccaa gaaacccgct aagaacctca
     3241 agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag gccaacaacg
     3301 agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc cctagcaaac
     3361 tggggcacaa gcttaattaa gaattcgtcg agggacctaa taacttcgta tagcatacat
     3421 tatacgaagt tatacatgtt taagggttcc ggttccacta ggtacaattc gatatcaagc
     3481 ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3541 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3601 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3661 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3721 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3781 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3841 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     3901 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3961 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     4021 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     4081 gataccgtcg acctcgatcg agacctagaa aaacatggag caatcacaag tagcaataca
     4141 gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt
     4201 ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
     4261 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     4321 atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca
     4381 ccagggccag ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt
     4441 gagcaagaga aggtagaaga agccaatgaa ggagagaaca cccgcttgtt acaccctgtg
     4501 agcctgcatg ggatggatga cccggagaga gaagtattag agtggaggtt tgacagccgc
     4561 ctagcatttc atcacatggc ccgagagctg catccggact gtactgggtc tctctggtta
     4621 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     4681 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     4741 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa
     4801 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4861 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4921 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4981 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5041 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5101 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5161 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5221 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5281 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5341 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5401 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5461 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5521 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5581 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5641 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5701 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5761 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5821 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5881 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5941 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6001 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6061 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6121 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6181 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6241 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6301 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6361 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6421 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6481 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6541 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6601 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     6661 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     6721 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     6781 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
     6841 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     6901 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     6961 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     7021 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     7081 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     7141 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     7201 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     7261 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     7321 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     7381 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
     7441 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     7501 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     7561 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     7621 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     7681 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     7741 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     7801 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     7861 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     7921 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     7981 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     8041 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     8101 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     8161 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     8221 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     8281 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     8341 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     8401 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     8461 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     8521 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     8581 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     8641 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     8701 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     8761 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     8821 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattc
//
