LOCUS       sequence-273586-        8877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CENPB CRISPRi guide RNA 4.
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-g4
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    146..263
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(516..549)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     553..573
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        818..1029
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1030..1968
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1925..1945
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1989..2585
                     /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(1989..2008)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2485..2505
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2595..2648
                     /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             2661..3359
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(3379..3412)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3468..4056
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3521..3541)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3939..3950)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4059..4075)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4060..4076)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4585..4765
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4827..5415)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4907..4926)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5586..6377,6378..6446))
                     /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     6209..6228
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6447..6551)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6626..6645)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6817..7196
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7198..7396
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7351..7371
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             7414..7594
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7641..7766
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8263..8496
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8681..8725
                     /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 cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag
       61 agacagagac agatccattc gattagtgaa cggatcggca ctgcgtgcgc caattctgca
      121 gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg
      181 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac
      241 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccagtttggt
      301 tagtaccggg cccgctctag agatccgacg cgccatctct aggcccgcgc cggccccctc
      361 gcacggactt gtgggagaag ctcggctact cccctgcccc ggttaatttg catataatat
      421 ttcctagtaa ctatagaggc ttaatgtgcg ataaaagaca gataatctgt tctttttaat
      481 actagctaca ttttacatga taggcttgga tttctataac ttcgtatagc atacattata
      541 cgaagttata aacagcacaa aaggaaactc accctaactg taaagtaatt gtgtgttttg
      601 agactataag tatcccttgg agaaccacct tgttggccgc cggcctgccg gtcaagttta
      661 agagctaagc tggaaacagc atagcaagtt taaataaggc tagtccgtta tcaacttgaa
      721 aaagtggcac cgagtcggtg ctttttttct cgagtactag gatccattag gcggccgcgt
      781 ggataaccgt attaccgcca tgcatgtgcc cgtcagtggg cagagcgcac atcgcccaca
      841 gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc
      901 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
      961 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     1021 agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc
     1081 ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt
     1141 cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt
     1201 gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt
     1261 cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct
     1321 gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt
     1381 atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg
     1441 gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc
     1501 cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg
     1561 ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca
     1621 gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa
     1681 aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg
     1741 ccgtccaggc acctcgatta gttctcgcgc ttttggagta cgtcgtcttt aggttggggg
     1801 gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca
     1861 gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc
     1921 attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgagct
     1981 agcccaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc
     2041 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     2101 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     2161 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     2221 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     2281 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     2341 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     2401 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     2461 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     2521 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     2581 gtgccggatc gggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc
     2641 ctggcccacc ggtcgccacc agcgagctga ttaaggagaa catgcacatg aagctgtaca
     2701 tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct
     2761 acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct
     2821 tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg
     2881 gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca
     2941 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
     3001 tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga
     3061 agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg
     3121 aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacatca
     3181 agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg
     3241 tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg
     3301 aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattaag
     3361 aattcgtcga gggacctaat aacttcgtat agcatacatt atacgaagtt atacatgttt
     3421 aagggttccg gttccactag gtacaattcg atatcaagct tatcgataat caacctctgg
     3481 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     3541 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     3601 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     3661 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     3721 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     3781 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     3841 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
     3901 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     3961 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     4021 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga cctcgatcga
     4081 gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa tgctgattgt
     4141 gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc tcaggtacct
     4201 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     4261 ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct gtggatctac
     4321 cacacacaag gctacttccc tgattggcag aactacacac cagggccagg gatcagatat
     4381 ccactgacct ttggatggtg ctacaagcta gtaccagttg agcaagagaa ggtagaagaa
     4441 gccaatgaag gagagaacac ccgcttgtta caccctgtga gcctgcatgg gatggatgac
     4501 ccggagagag aagtattaga gtggaggttt gacagccgcc tagcatttca tcacatggcc
     4561 cgagagctgc atccggactg tactgggtct ctctggttag accagatctg agcctgggag
     4621 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     4681 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     4741 tagtcagtgt ggaaaatctc tagcagcatg tgagcaaaag gccagcaaaa ggccaggaac
     4801 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4861 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4921 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4981 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5041 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5101 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5161 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5221 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5281 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5341 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5401 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5461 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5521 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5581 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5641 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5701 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5761 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5821 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5881 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5941 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6001 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6061 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6121 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6181 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6241 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6301 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6361 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6421 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6481 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6541 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     6601 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     6661 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     6721 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
     6781 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
     6841 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     6901 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     6961 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     7021 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     7081 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     7141 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     7201 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     7261 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     7321 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     7381 aggtctatat aagcagcgcg ttttgcctgt actgggtctc tctggttaga ccagatctga
     7441 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     7501 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     7561 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
     7621 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
     7681 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
     7741 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
     7801 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
     7861 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
     7921 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     7981 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     8041 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     8101 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     8161 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     8221 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     8281 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     8341 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     8401 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     8461 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     8521 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     8581 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     8641 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     8701 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     8761 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     8821 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agaccca
//
