LOCUS       sequence-273585-        8877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CENPB CRISPRi guide RNA 3.
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-g3
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    126..243
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(496..529)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     533..553
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        798..1009
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1010..1948
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1905..1925
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1969..2565
                     /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(1969..1988)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2465..2485
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2575..2628
                     /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             2641..3339
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(3359..3392)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3448..4036
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3501..3521)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3919..3930)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4039..4055)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4040..4056)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4565..4745
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4807..5395)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4887..4906)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5566..6357,6358..6426))
                     /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     6189..6208
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6427..6531)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6606..6625)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6797..7176
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7178..7376
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7331..7351
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             7394..7574
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7621..7746
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8243..8476
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8661..8705
                     /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 ccgacaggcc cgaaggaata gaagaagaag gtggagagag agacagagac agatccattc
       61 gattagtgaa cggatcggca ctgcgtgcgc caattctgca gacaaatggc agtattcatc
      121 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
      181 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
      241 tttcgggttt attacaggga cagcagagat ccagtttggt tagtaccggg cccgctctag
      301 agatccgacg cgccatctct aggcccgcgc cggccccctc gcacggactt gtgggagaag
      361 ctcggctact cccctgcccc ggttaatttg catataatat ttcctagtaa ctatagaggc
      421 ttaatgtgcg ataaaagaca gataatctgt tctttttaat actagctaca ttttacatga
      481 taggcttgga tttctataac ttcgtatagc atacattata cgaagttata aacagcacaa
      541 aaggaaactc accctaactg taaagtaatt gtgtgttttg agactataag tatcccttgg
      601 agaaccacct tgttggagaa tccggacctg cgcaagttta agagctaagc tggaaacagc
      661 atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      721 ctttttttct cgagtactag gatccattag gcggccgcgt ggataaccgt attaccgcca
      781 tgcatgtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
      841 aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
      901 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
      961 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt
     1021 gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac
     1081 ttccacctgg ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga
     1141 gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc
     1201 tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt
     1261 cgataagtct ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca
     1321 agatagtctt gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc
     1381 gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc
     1441 gcggccaccg agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg
     1501 cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt
     1561 tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac
     1621 gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc
     1681 ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta
     1741 gttctcgcgc ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga
     1801 gtttccccac actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt
     1861 ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt
     1921 ggttcaaagt ttttttcttc catttcaggt gtcgtgagct agcccaccat gaccgagtac
     1981 aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg caccctcgcc
     2041 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg acccggaccg ccacatcgag
     2101 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     2161 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     2221 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     2281 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     2341 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     2401 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     2461 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     2521 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgccggatc gggagagggc
     2581 agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccacc ggtcgccacc
     2641 agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac cgtggacaac
     2701 catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg
     2761 agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct ggctactagc
     2821 ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga cttcttcaag
     2881 cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga cgggggcgtg
     2941 ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaagatc
     3001 agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact cggctgggag
     3061 gccttcaccg agacgctgta ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc
     3121 ctgaagctcg tgggcgggag ccatctgatc gcaaacatca agaccacata tagatccaag
     3181 aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag actggaaaga
     3241 atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt ggccagatac
     3301 tgcgacctcc ctagcaaact ggggcacaag cttaattaag aattcgtcga gggacctaat
     3361 aacttcgtat agcatacatt atacgaagtt atacatgttt aagggttccg gttccactag
     3421 gtacaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga
     3481 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     3541 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     3601 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     3661 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     3721 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     3781 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     3841 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     3901 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     3961 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     4021 tgggccgcct ccccgcatcg ataccgtcga cctcgatcga gacctagaaa aacatggagc
     4081 aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga
     4141 ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa
     4201 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     4261 ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag gctacttccc
     4321 tgattggcag aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg
     4381 ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac
     4441 ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga
     4501 gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg
     4561 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     4621 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     4681 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     4741 tagcagcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4801 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4861 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4921 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4981 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5041 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5101 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5161 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5221 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5281 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5341 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5401 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5461 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5521 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5581 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5641 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5701 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5761 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5821 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5881 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5941 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6001 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6061 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6121 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6181 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6241 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6301 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6361 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6421 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6481 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6541 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
     6601 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
     6661 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
     6721 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
     6781 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
     6841 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     6901 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     6961 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     7021 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     7081 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     7141 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     7201 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     7261 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     7321 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg
     7381 ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     7441 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     7501 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     7561 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
     7621 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
     7681 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
     7741 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
     7801 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
     7861 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
     7921 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
     7981 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     8041 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgctgatct
     8101 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag
     8161 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
     8221 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
     8281 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta
     8341 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
     8401 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa
     8461 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
     8521 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct
     8581 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
     8641 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
     8701 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga
     8761 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag
     8821 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggac
//
