LOCUS       sequence-227843-        8888 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 8888)
  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 8888)
  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..8888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        182..393
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          394..1332
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1289..1309
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1353..1949
                     /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(1353..1372)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1849..1869
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1959..2012
                     /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             2025..2723
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(2743..2776)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2832..3420
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2885..2905)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3303..3314)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3423..3439)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3424..3440)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             3949..4129
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4191..4779)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4271..4290)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4950..5741,5742..5810))
                     /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     5573..5592
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5811..5915)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5999..6018)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6190..6569
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6571..6769
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6724..6744
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6787..6967
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7014..7139
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7638..7871
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8056..8100
                     /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    8398..8515
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(8768..8801)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     8805..8825
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
ORIGIN
        1 ccgccggcct gccggtcaag tttaagagct aagctggaaa cagcatagca agtttaaata
       61 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctcgagta
      121 ctaggatcca ttaggcggcc gcgtggataa ccgtattacc gccatgcatg tgcccgtcag
      181 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
      241 accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
      301 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
      361 ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg
      421 cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctgcag
      481 tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc
      541 gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc
      601 cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc
      661 atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat
      721 gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc
      781 cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc
      841 ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt
      901 atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga
      961 tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag
     1021 cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca
     1081 tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc gcgcttttgg
     1141 agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag
     1201 tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc
     1261 ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt
     1321 cttccatttc aggtgtcgtg agctagccca ccatgaccga gtacaagccc acggtgcgcc
     1381 tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg ttcgccgact
     1441 accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc
     1501 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     1561 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     1621 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     1681 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     1741 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     1801 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     1861 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     1921 cctggtgcat gacccgcaag cccggtgccg gatcgggaga gggcagagga agtctgctaa
     1981 catgcggtga cgtcgaggag aatcctggcc caccggtcgc caccagcgag ctgattaagg
     2041 agaacatgca catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca
     2101 catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg
     2161 agggcggccc tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca
     2221 agaccttcat caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg
     2281 gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg
     2341 acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca
     2401 catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc
     2461 tgtaccccgc tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg
     2521 ggagccatct gatcgcaaac atcaagacca catatagatc caagaaaccc gctaagaacc
     2581 tcaagatgcc tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca
     2641 acgagaccta cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca
     2701 aactggggca caagcttaat taagaattcg tcgagggacc taataacttc gtatagcata
     2761 cattatacga agttatacat gtttaagggt tccggttcca ctaggtacaa ttcgatatca
     2821 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     2881 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     2941 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     3001 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     3061 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     3121 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     3181 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     3241 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     3301 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     3361 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     3421 atcgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac aagtagcaat
     3481 acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt
     3541 tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt
     3601 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     3661 gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg gcagaactac
     3721 acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa gctagtacca
     3781 gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt gttacaccct
     3841 gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag gtttgacagc
     3901 cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg gtctctctgg
     3961 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     4021 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     4081 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag catgtgagca
     4141 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4201 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4261 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4321 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4381 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4441 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4501 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4561 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4621 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4681 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4741 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4801 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4861 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4921 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4981 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5041 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5101 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5161 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5221 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5281 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5341 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5401 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5461 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5521 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5581 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5641 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5701 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5761 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5821 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5881 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5941 gacgcttaag cggtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta
     6001 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     6061 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     6121 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     6181 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     6241 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     6301 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     6361 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     6421 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     6481 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     6541 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     6601 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     6661 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     6721 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcagc gcgttttgcc
     6781 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     6841 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     6901 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     6961 tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc
     7021 gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga
     7081 gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca
     7141 gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa
     7201 agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag
     7261 ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac
     7321 catcccttca gacaggatca gaagaactta gatcattata taatacagta gcaaccctct
     7381 attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg
     7441 aagagcaaaa caaaagtaag accaccgcac agcaagcggc cggccgcgct gatcttcaga
     7501 cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
     7561 aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa
     7621 aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact
     7681 atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg
     7741 cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca
     7801 gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
     7861 caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct
     7921 tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg
     7981 gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg
     8041 caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg
     8101 tggaattggt ttaacataac aaattggctg tggtatataa aattattcat aatgatagta
     8161 ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg
     8221 cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg
     8281 cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg
     8341 aacggatcgg cactgcgtgc gccaattctg cagacaaatg gcagtattca tccacaattt
     8401 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     8461 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     8521 ttattacagg gacagcagag atccagtttg gttagtaccg ggcccgctct agagatccga
     8581 cgcgccatct ctaggcccgc gccggccccc tcgcacggac ttgtgggaga agctcggcta
     8641 ctcccctgcc ccggttaatt tgcatataat atttcctagt aactatagag gcttaatgtg
     8701 cgataaaaga cagataatct gttcttttta atactagcta cattttacat gataggcttg
     8761 gatttctata acttcgtata gcatacatta tacgaagtta taaacagcac aaaaggaaac
     8821 tcaccctaac tgtaaagtaa ttgtgtgttt tgagactata agtatccctt ggagaaccac
     8881 cttgttgg
//
