LOCUS       sequence-273601-        8877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CENPB CRISPRi guide RNA 8.
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-g8
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    189..306
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(559..592)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     596..616
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        861..1072
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1073..2011
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1968..1988
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2032..2628
                     /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(2032..2051)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2528..2548
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2638..2691
                     /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             2704..3402
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(3422..3455)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3511..4099
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3564..3584)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3982..3993)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4102..4118)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4103..4119)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4628..4808
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4870..5458)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4950..4969)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5629..6420,6421..6489))
                     /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     6252..6271
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6490..6594)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6669..6688)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6860..7239
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7241..7439
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7394..7414
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             7457..7637
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7684..7809
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8306..8539
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8724..8768
                     /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 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
       61 gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca
      121 ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc
      181 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
      241 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
      301 aattttcggg tttattacag ggacagcaga gatccagttt ggttagtacc gggcccgctc
      361 tagagatccg acgcgccatc tctaggcccg cgccggcccc ctcgcacgga cttgtgggag
      421 aagctcggct actcccctgc cccggttaat ttgcatataa tatttcctag taactataga
      481 ggcttaatgt gcgataaaag acagataatc tgttcttttt aatactagct acattttaca
      541 tgataggctt ggatttctat aacttcgtat agcatacatt atacgaagtt ataaacagca
      601 caaaaggaaa ctcaccctaa ctgtaaagta attgtgtgtt ttgagactat aagtatccct
      661 tggagaacca ccttgttggg cgcaagggcg agatcgcggt ttaagagcta agctggaaac
      721 agcatagcaa gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
      781 gtgctttttt tctcgagtac taggatccat taggcggccg cgtggataac cgtattaccg
      841 ccatgcatgt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      901 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      961 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
     1021 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
     1081 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
     1141 tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg
     1201 ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg
     1261 gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc
     1321 tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg
     1381 gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc
     1441 cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg
     1501 agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc
     1561 tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc
     1621 agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag
     1681 gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc
     1741 gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga
     1801 ttagttctcg cgcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat
     1861 ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta
     1921 attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac
     1981 agtggttcaa agtttttttc ttccatttca ggtgtcgtga gctagcccac catgaccgag
     2041 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc cccgggccgt acgcaccctc
     2101 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgacccgga ccgccacatc
     2161 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     2221 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     2281 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     2341 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     2401 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     2461 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     2521 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     2581 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg atcgggagag
     2641 ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc accggtcgcc
     2701 accagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac
     2761 aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc
     2821 atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact
     2881 agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc
     2941 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
     3001 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag
     3061 atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg
     3121 gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg
     3181 gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac atatagatcc
     3241 aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa
     3301 agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga
     3361 tactgcgacc tccctagcaa actggggcac aagcttaatt aagaattcgt cgagggacct
     3421 aataacttcg tatagcatac attatacgaa gttatacatg tttaagggtt ccggttccac
     3481 taggtacaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     3541 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     3601 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3661 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3721 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3781 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     3841 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     3901 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     3961 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     4021 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     4081 ctttgggccg cctccccgca tcgataccgt cgacctcgat cgagacctag aaaaacatgg
     4141 agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc tagaagcaca
     4201 agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac caatgactta
     4261 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     4321 tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac aaggctactt
     4381 ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga cctttggatg
     4441 gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa
     4501 cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtatt
     4561 agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc tgcatccgga
     4621 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     4681 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     4741 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     4801 ctctagcagc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4861 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4921 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4981 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5041 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5101 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5161 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5221 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5281 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5341 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5401 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5461 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5521 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5581 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5641 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5701 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5761 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5821 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5881 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5941 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6001 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6061 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6121 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6181 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6241 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6301 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6361 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6421 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6481 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6541 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6601 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
     6661 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
     6721 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     6781 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     6841 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
     6901 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     6961 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     7021 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     7081 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     7141 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     7201 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     7261 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     7321 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     7381 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcagc
     7441 gcgttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     7501 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     7561 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     7621 gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag
     7681 gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg
     7741 cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc
     7801 gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg
     7861 ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa
     7921 cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga
     7981 cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta
     8041 gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac
     8101 aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cggccgctga
     8161 tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata
     8221 aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc
     8281 agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag
     8341 gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg
     8401 gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc
     8461 aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc
     8521 taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg
     8581 ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga
     8641 cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg
     8701 aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg
     8761 caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa
     8821 tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtga
//
