LOCUS       sequence-273588-        8877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CENPB CRISPRi guide RNA 5.
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-g5
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    122..355
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             540..584
                     /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    882..999
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(1252..1285)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1289..1309
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        1554..1765
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1766..2704
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2661..2681
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2725..3321
                     /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(2725..2744)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3221..3241
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3331..3384
                     /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             3397..4095
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(4115..4148)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4204..4792
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4257..4277)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4675..4686)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4795..4811)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4796..4812)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5321..5501
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5563..6151)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5643..5662)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6322..7113,7114..7182))
                     /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     6945..6964
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7183..7287)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7362..7381)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7553..7932
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7934..8132
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8087..8107
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             8150..8330
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    8377..8502
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
ORIGIN
        1 tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag
       61 gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa
      121 taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa
      181 tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt
      241 tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc
      301 agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga
      361 tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga
      421 gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa
      481 ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa
      541 agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca
      601 taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt
      661 taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat
      721 tatcgtttca gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag
      781 aagaaggtgg agagagagac agagacagat ccattcgatt agtgaacgga tcggcactgc
      841 gtgcgccaat tctgcagaca aatggcagta ttcatccaca attttaaaag aaaagggggg
      901 attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact
      961 aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc
     1021 agagatccag tttggttagt accgggcccg ctctagagat ccgacgcgcc atctctaggc
     1081 ccgcgccggc cccctcgcac ggacttgtgg gagaagctcg gctactcccc tgccccggtt
     1141 aatttgcata taatatttcc tagtaactat agaggcttaa tgtgcgataa aagacagata
     1201 atctgttctt tttaatacta gctacatttt acatgatagg cttggatttc tataacttcg
     1261 tatagcatac attatacgaa gttataaaca gcacaaaagg aaactcaccc taactgtaaa
     1321 gtaattgtgt gttttgagac tataagtatc ccttggagaa ccaccttgtt ggcggccgcg
     1381 gtgccctcgg agtttaagag ctaagctgga aacagcatag caagtttaaa taaggctagt
     1441 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttctcgag tactaggatc
     1501 cattaggcgg ccgcgtggat aaccgtatta ccgccatgca tgtgcccgtc agtgggcaga
     1561 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc
     1621 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
     1681 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     1741 caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct
     1801 ctttacgggt tatggccctt gcgtgccttg aattacttcc acctggctgc agtacgtgat
     1861 tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg
     1921 agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg
     1981 aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa
     2041 tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca
     2101 agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc
     2161 ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg
     2221 gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc
     2281 gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct
     2341 tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg
     2401 tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc
     2461 cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgcgctttt ggagtacgtc
     2521 gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga
     2581 gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga
     2641 gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt
     2701 tcaggtgtcg tgagctagcc caccatgacc gagtacaagc ccacggtgcg cctcgccacc
     2761 cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     2821 acgcgccaca ccgtcgaccc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     2881 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     2941 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     3001 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     3061 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     3121 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     3181 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     3241 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     3301 atgacccgca agcccggtgc cggatcggga gagggcagag gaagtctgct aacatgcggt
     3361 gacgtcgagg agaatcctgg cccaccggtc gccaccagcg agctgattaa ggagaacatg
     3421 cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg cacatccgag
     3481 ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc
     3541 cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag caagaccttc
     3601 atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga gggcttcaca
     3661 tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc
     3721 ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt cacatccaac
     3781 ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgagac gctgtacccc
     3841 gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg cgggagccat
     3901 ctgatcgcaa acatcaagac cacatataga tccaagaaac ccgctaagaa cctcaagatg
     3961 cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa caacgagacc
     4021 tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag caaactgggg
     4081 cacaagctta attaagaatt cgtcgaggga cctaataact tcgtatagca tacattatac
     4141 gaagttatac atgtttaagg gttccggttc cactaggtac aattcgatat caagcttatc
     4201 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     4261 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     4321 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     4381 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     4441 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     4501 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     4561 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     4621 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     4681 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     4741 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     4801 cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc acaagtagca atacagcagc
     4861 taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt
     4921 cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     4981 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct
     5041 tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact acacaccagg
     5101 gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac cagttgagca
     5161 agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc ctgtgagcct
     5221 gcatgggatg gatgacccgg agagagaagt attagagtgg aggtttgaca gccgcctagc
     5281 atttcatcac atggcccgag agctgcatcc ggactgtact gggtctctct ggttagacca
     5341 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     5401 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     5461 atccctcaga cccttttagt cagtgtggaa aatctctagc agcatgtgag caaaaggcca
     5521 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5581 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5641 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5701 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5761 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5821 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5881 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5941 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6001 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6061 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6121 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6181 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6241 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6301 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6361 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6421 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6481 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6541 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6601 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6661 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6721 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6781 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6841 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6901 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6961 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7021 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7081 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7141 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7201 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7261 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     7321 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     7381 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     7441 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     7501 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga
     7561 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     7621 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     7681 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     7741 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     7801 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     7861 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     7921 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     7981 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     8041 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     8101 aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt gcctgtactg ggtctctctg
     8161 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     8221 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     8281 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
     8341 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
     8401 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg
     8461 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
     8521 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta
     8581 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
     8641 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
     8701 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
     8761 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt
     8821 aagaccaccg cacagcaagc ggccggccgc tgatcttcag acctggagga ggagata
//
