LOCUS       sequence-273662-        8877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CENPB CRISPRi guide RNA 9.
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-g9
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    159..392
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             577..621
                     /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    919..1036
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(1289..1322)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1326..1346
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        1591..1802
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1803..2741
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2698..2718
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2762..3358
                     /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(2762..2781)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3258..3278
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3368..3421
                     /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             3434..4132
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(4152..4185)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4241..4829
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4294..4314)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4712..4723)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4832..4848)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4833..4849)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5358..5538
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5600..6188)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5680..5699)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6359..7150,7151..7219))
                     /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     6982..7001
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7220..7324)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7399..7418)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7590..7969
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7971..8169
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8124..8144
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             8187..8367
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    8414..8539
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
ORIGIN
        1 ggccggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa
       61 ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag
      121 agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc
      181 ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga
      241 caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa
      301 cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct
      361 gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc
      421 atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt
      481 tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata
      541 cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa
      601 ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata
      661 aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt
      721 tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca
      781 accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga
      841 gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat
      901 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga
      961 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac
     1021 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt ggttagtacc
     1081 gggcccgctc tagagatccg acgcgccatc tctaggcccg cgccggcccc ctcgcacgga
     1141 cttgtgggag aagctcggct actcccctgc cccggttaat ttgcatataa tatttcctag
     1201 taactataga ggcttaatgt gcgataaaag acagataatc tgttcttttt aatactagct
     1261 acattttaca tgataggctt ggatttctat aacttcgtat agcatacatt atacgaagtt
     1321 ataaacagca caaaaggaaa ctcaccctaa ctgtaaagta attgtgtgtt ttgagactat
     1381 aagtatccct tggagaacca ccttgttggc tccctacgac aagctcgagt ttaagagcta
     1441 agctggaaac agcatagcaa gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg
     1501 caccgagtcg gtgctttttt tctcgagtac taggatccat taggcggccg cgtggataac
     1561 cgtattaccg ccatgcatgt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
     1621 agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa
     1681 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
     1741 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
     1801 aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg
     1861 tgccttgaat tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg
     1921 gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag
     1981 ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct
     2041 gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc
     2101 tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg
     2161 tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc
     2221 ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg
     2281 ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc
     2341 ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct
     2401 caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa
     2461 gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca
     2521 ggcacctcga ttagttctcg cgcttttgga gtacgtcgtc tttaggttgg ggggaggggt
     2581 tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc
     2641 acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca
     2701 agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga gctagcccac
     2761 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc cccgggccgt
     2821 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgacccgga
     2881 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     2941 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     3001 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     3061 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     3121 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     3181 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     3241 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     3301 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg
     3361 atcgggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc
     3421 accggtcgcc accagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
     3481 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
     3541 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
     3601 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc
     3661 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     3721 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     3781 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac
     3841 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag
     3901 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac
     3961 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta
     4021 cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc
     4081 agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatt aagaattcgt
     4141 cgagggacct aataacttcg tatagcatac attatacgaa gttatacatg tttaagggtt
     4201 ccggttccac taggtacaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa
     4261 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     4321 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     4381 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     4441 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     4501 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     4561 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     4621 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     4681 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     4741 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     4801 tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgat cgagacctag
     4861 aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc
     4921 tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac
     4981 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     5041 aagggctaat tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac
     5101 aaggctactt ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga
     5161 cctttggatg gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg
     5221 aaggagagaa cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga
     5281 gagaagtatt agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc
     5341 tgcatccgga ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     5401 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     5461 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     5521 tgtggaaaat ctctagcagc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5581 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5641 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5701 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5761 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5821 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5881 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5941 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6001 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6061 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6121 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6181 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6241 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6301 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6361 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6421 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6481 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6541 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6601 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6661 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6721 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6781 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6841 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6901 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6961 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7021 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7081 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7141 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7201 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7261 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7321 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     7381 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     7441 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     7501 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     7561 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     7621 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     7681 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     7741 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     7801 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     7861 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     7921 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     7981 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     8041 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     8101 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     8161 tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg
     8221 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     8281 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     8341 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
     8401 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
     8461 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
     8521 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
     8581 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
     8641 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
     8701 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
     8761 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
     8821 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagc
//
