LOCUS       sequence-273583-        8877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CENPB CRISPRi guide RNA 2.
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-g2
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    64..181
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(434..467)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     471..491
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        736..947
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          948..1886
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1843..1863
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1907..2503
                     /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(1907..1926)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2403..2423
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2513..2566
                     /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             2579..3277
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(3297..3330)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3386..3974
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3439..3459)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3857..3868)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3977..3993)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3978..3994)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4503..4683
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4745..5333)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4825..4844)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5504..6295,6296..6364))
                     /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     6127..6146
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6365..6469)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6544..6563)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6735..7114
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7116..7314
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7269..7289
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             7332..7512
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7559..7684
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8181..8414
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8599..8643
                     /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 ttagtgaacg gatcggcact gcgtgcgcca attctgcaga caaatggcag tattcatcca
       61 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
      121 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
      181 tcgggtttat tacagggaca gcagagatcc agtttggtta gtaccgggcc cgctctagag
      241 atccgacgcg ccatctctag gcccgcgccg gccccctcgc acggacttgt gggagaagct
      301 cggctactcc cctgccccgg ttaatttgca tataatattt cctagtaact atagaggctt
      361 aatgtgcgat aaaagacaga taatctgttc tttttaatac tagctacatt ttacatgata
      421 ggcttggatt tctataactt cgtatagcat acattatacg aagttataaa cagcacaaaa
      481 ggaaactcac cctaactgta aagtaattgt gtgttttgag actataagta tcccttggag
      541 aaccaccttg ttggtcggag cgcaagtacg ggggtttaag agctaagctg gaaacagcat
      601 agcaagttta aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
      661 ttttttctcg agtactagga tccattaggc ggccgcgtgg ataaccgtat taccgccatg
      721 catgtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
      781 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
      841 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
      901 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt
      961 gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt
     1021 ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga
     1081 gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg
     1141 ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg
     1201 ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag
     1261 atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg
     1321 gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc
     1381 ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc
     1441 tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg
     1501 cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc
     1561 ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct
     1621 cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt
     1681 tctcgcgctt ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt
     1741 ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct
     1801 ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg
     1861 ttcaaagttt ttttcttcca tttcaggtgt cgtgagctag cccaccatga ccgagtacaa
     1921 gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc
     1981 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg
     2041 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     2101 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     2161 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     2221 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     2281 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     2341 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc
     2401 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     2461 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gccggatcgg gagagggcag
     2521 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaccgg tcgccaccag
     2581 cgagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tggacaacca
     2641 tcacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag
     2701 aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctactagctt
     2761 cctctacggc agcaagacct tcatcaacca cacccagggc atccccgact tcttcaagca
     2821 gtccttccct gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct
     2881 gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag
     2941 aggggtgaac ttcacatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc
     3001 cttcaccgag acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg acatggccct
     3061 gaagctcgtg ggcgggagcc atctgatcgc aaacatcaag accacatata gatccaagaa
     3121 acccgctaag aacctcaaga tgcctggcgt ctactatgtg gactacagac tggaaagaat
     3181 caaggaggcc aacaacgaga cctacgtcga gcagcacgag gtggcagtgg ccagatactg
     3241 cgacctccct agcaaactgg ggcacaagct taattaagaa ttcgtcgagg gacctaataa
     3301 cttcgtatag catacattat acgaagttat acatgtttaa gggttccggt tccactaggt
     3361 acaattcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     3421 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     3481 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     3541 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     3601 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     3661 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     3721 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     3781 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     3841 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     3901 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     3961 ggccgcctcc ccgcatcgat accgtcgacc tcgatcgaga cctagaaaaa catggagcaa
     4021 tcacaagtag caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg
     4081 aggaggaggt gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg
     4141 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     4201 cccaacgaag acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg
     4261 attggcagaa ctacacacca gggccaggga tcagatatcc actgaccttt ggatggtgct
     4321 acaagctagt accagttgag caagagaagg tagaagaagc caatgaagga gagaacaccc
     4381 gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtattagagt
     4441 ggaggtttga cagccgccta gcatttcatc acatggcccg agagctgcat ccggactgta
     4501 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     4561 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     4621 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     4681 gcagcatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4741 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4801 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4861 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4921 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4981 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5041 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5101 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5161 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5221 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5281 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5341 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5401 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5461 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5521 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5581 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5641 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5701 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5761 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5821 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5881 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5941 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6001 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6061 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6121 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6181 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6241 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6301 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6361 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6421 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6481 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     6541 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     6601 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     6661 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     6721 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
     6781 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     6841 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     6901 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     6961 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     7021 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     7081 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
     7141 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
     7201 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     7261 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagcgcgtt
     7321 ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     7381 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     7441 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     7501 aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct
     7561 ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact
     7621 ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag
     7681 cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg
     7741 gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt
     7801 cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct
     7861 acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac
     7921 cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat
     7981 agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc gctgatcttc
     8041 agacctggag gaggagatat gagggacaat tggagaagtg aattatataa atataaagta
     8101 gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga
     8161 gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc
     8221 actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata
     8281 gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc
     8341 acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag
     8401 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg
     8461 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg
     8521 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa
     8581 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt
     8641 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata
     8701 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt
     8761 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac
     8821 aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcga
//
