LOCUS       sequence-273579-        8877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CENPB CRISPRi guide RNA 1.
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-g1
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(208..241)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     245..265
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        510..721
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          722..1660
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1617..1637
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1681..2277
                     /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(1681..1700)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2177..2197
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2287..2340
                     /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             2353..3051
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(3071..3104)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3160..3748
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3213..3233)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3631..3642)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3751..3767)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3752..3768)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4277..4457
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4519..5107)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4599..4618)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5278..6069,6070..6138))
                     /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     5901..5920
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6139..6243)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6318..6337)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6509..6888
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6890..7088
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7043..7063
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             7106..7286
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7333..7458
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7955..8188
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8373..8417
                     /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    8715..8832
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 ggcccgctct agagatccga cgcgccatct ctaggcccgc gccggccccc tcgcacggac
       61 ttgtgggaga agctcggcta ctcccctgcc ccggttaatt tgcatataat atttcctagt
      121 aactatagag gcttaatgtg cgataaaaga cagataatct gttcttttta atactagcta
      181 cattttacat gataggcttg gatttctata acttcgtata gcatacatta tacgaagtta
      241 taaacagcac aaaaggaaac tcaccctaac tgtaaagtaa ttgtgtgttt tgagactata
      301 agtatccctt ggagaaccac cttgttgggc gtcggagcgc aagtacggtt taagagctaa
      361 gctggaaaca gcatagcaag tttaaataag gctagtccgt tatcaacttg aaaaagtggc
      421 accgagtcgg tgcttttttt ctcgagtact aggatccatt aggcggccgc gtggataacc
      481 gtattaccgc catgcatgtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
      541 gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa
      601 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
      661 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
      721 ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt
      781 gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg
      841 aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt
      901 tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg
      961 tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct
     1021 ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt
     1081 ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg
     1141 gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc
     1201 tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg
     1261 gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc
     1321 aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag
     1381 ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag
     1441 gcacctcgat tagttctcgc gcttttggag tacgtcgtct ttaggttggg gggaggggtt
     1501 ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca
     1561 cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa
     1621 gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgag ctagcccacc
     1681 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta
     1741 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac
     1801 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     1861 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     1921 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     1981 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     2041 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     2101 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     2161 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     2221 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgccgga
     2281 tcgggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccca
     2341 ccggtcgcca ccagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc
     2401 accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
     2461 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
     2521 ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc
     2581 gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa
     2641 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
     2701 aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca
     2761 ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga
     2821 aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacat caagaccaca
     2881 tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac
     2941 agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca
     3001 gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaatta agaattcgtc
     3061 gagggaccta ataacttcgt atagcataca ttatacgaag ttatacatgt ttaagggttc
     3121 cggttccact aggtacaatt cgatatcaag cttatcgata atcaacctct ggattacaaa
     3181 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     3241 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     3301 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     3361 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     3421 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     3481 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     3541 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     3601 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     3661 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     3721 cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgatc gagacctaga
     3781 aaaacatgga gcaatcacaa gtagcaatac agcagctacc aatgctgatt gtgcctggct
     3841 agaagcacaa gaggaggagg aggtgggttt tccagtcaca cctcaggtac ctttaagacc
     3901 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     3961 agggctaatt cactcccaac gaagacaaga tatccttgat ctgtggatct accacacaca
     4021 aggctacttc cctgattggc agaactacac accagggcca gggatcagat atccactgac
     4081 ctttggatgg tgctacaagc tagtaccagt tgagcaagag aaggtagaag aagccaatga
     4141 aggagagaac acccgcttgt tacaccctgt gagcctgcat gggatggatg acccggagag
     4201 agaagtatta gagtggaggt ttgacagccg cctagcattt catcacatgg cccgagagct
     4261 gcatccggac tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     4321 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     4381 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     4441 gtggaaaatc tctagcagca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4501 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4561 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4621 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4681 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4741 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4801 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4861 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4921 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4981 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5041 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5101 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5161 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5221 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5281 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5341 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5401 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5461 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5521 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5581 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5641 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5701 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5761 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5821 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5881 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5941 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6001 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6061 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6121 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6181 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6241 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     6301 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     6361 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     6421 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     6481 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
     6541 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     6601 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     6661 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     6721 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     6781 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     6841 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     6901 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     6961 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     7021 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     7081 ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga
     7141 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     7201 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     7261 ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg
     7321 aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc
     7381 gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga
     7441 gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt
     7501 cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg
     7561 gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa
     7621 atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat
     7681 aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa
     7741 gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc
     7801 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat
     7861 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa
     7921 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg
     7981 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat
     8041 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc
     8101 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg
     8161 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt
     8221 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga
     8281 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact
     8341 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag
     8401 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat
     8461 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta
     8521 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc
     8581 cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca
     8641 gatccattcg attagtgaac ggatcggcac tgcgtgcgcc aattctgcag acaaatggca
     8701 gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga
     8761 atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa
     8821 attcaaaatt ttcgggttta ttacagggac agcagagatc cagtttggtt agtaccg
//
