LOCUS       sequence-273663-        8877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CENPB CRISPRi guide RNA 10.
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-g10
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    19..607
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             125..136
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     534..554
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    663..696
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(716..1414)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             complement(1427..1480)
                     /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             complement(1490..2086)
                     /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(1570..1590)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2067..2086
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     intron          2107..3045
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2130..2150)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(3046..3257)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     primer_bind     complement(3502..3522)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    3526..3559
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3812..3929
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(4227..4271)
                     /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    4456..4689
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    5186..5311
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             5358..5538
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(5556..5754)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5581..5601)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5756..6135
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6307..6326
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6401..6505
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6506..6574,6575..7366)
                     /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     complement(6724..6743)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7537..8125
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8026..8045
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     LTR             8187..8367
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     join(8876..8877,1..15)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     join(8877,1..16)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gaggtcgacg gtatcgatgc ggggaggcgg cccaaaggga gatccgactc gtctgagggc
       61 gaaggcgaag acgcggaaga ggccgcagag ccggcagcag gccgcgggaa ggaaggtccg
      121 ctggattgag ggccgaaggg acgtagcaga aggacgtccc gcgcagaatc caggtggcaa
      181 cacaggcgag cagccaagga aaggacgatg atttccccga caacaccacg gaattgtcag
      241 tgcccaacag ccgagcccct gtccagcagc gggcaaggca ggcggcgatg agttccgccg
      301 tggcaatagg gagggggaaa gcgaaagtcc cggaaaggag ctgacaggtg gtggcaatgc
      361 cccaaccagt gggggttgcg tcagcaaaca cagtgcacac cacgccacgt tgcctgacaa
      421 cgggccacaa ctcctcataa agagacagca accaggattt atacaaggag gagaaaatga
      481 aagccatacg ggaagcaata gcatgataca aaggcattaa agcagcgtat ccacatagcg
      541 taaaaggagc aacatagtta agaataccag tcaatctttc acaaattttg taatccagag
      601 gttgattatc gataagcttg atatcgaatt gtacctagtg gaaccggaac ccttaaacat
      661 gtataacttc gtataatgta tgctatacga agttattagg tccctcgacg aattcttaat
      721 taagcttgtg ccccagtttg ctagggaggt cgcagtatct ggccactgcc acctcgtgct
      781 gctcgacgta ggtctcgttg ttggcctcct tgattctttc cagtctgtag tccacatagt
      841 agacgccagg catcttgagg ttcttagcgg gtttcttgga tctatatgtg gtcttgatgt
      901 ttgcgatcag atggctcccg cccacgagct tcagggccat gtcgtttctg ccttccaggc
      961 cgccgtcagc ggggtacagc gtctcggtga aggcctccca gccgagtgtt ttcttctgca
     1021 tcacagggcc gttggatgtg aagttcaccc ctctgatctt gacgttgtag atgaggcagc
     1081 cgtcctggag gctggtgtcc tgggtagcgg tcagcacgcc cccgtcttcg tatgtggtga
     1141 ctctctccca tgtgaagccc tcagggaagg actgcttgaa gaagtcgggg atgccctggg
     1201 tgtggttgat gaaggtcttg ctgccgtaga ggaagctagt agccaggatg tcgaaggcga
     1261 aggggagagg gccgccctcg accaccttga ttctcatggt ctgggtgccc tcgtagggct
     1321 tgccttcgcc ctcggatgtg cacttgaagt gatggttgtc cacggtgccc tccatgtaca
     1381 gcttcatgtg catgttctcc ttaatcagct cgctggtggc gaccggtggg ccaggattct
     1441 cctcgacgtc accgcatgtt agcagacttc ctctgccctc tcccgatccg gcaccgggct
     1501 tgcgggtcat gcaccaggtg cgcggtcctt cgggcacctc gacgtcggcg gtgacggtga
     1561 agccgagccg ctcgtagaag gggaggttgc ggggcgcgga ggtctccagg aaggcgggca
     1621 ccccggcgcg ctcggccgcc tccactccgg ggagcacgac ggcgctgccc agacccttgc
     1681 cctggtggtc gggcgagacg ccgacggtgg ccaggaacca cgcgggctcc ttgggccggt
     1741 gcggcgccag gaggccttcc atctgttgct gcgcggccag ccgggaaccg ctcaactcgg
     1801 ccatgcgcgg gccgatctcg gcgaacaccg cccccgcttc gacgctctcc ggcgtggtcc
     1861 agaccgccac cgcggcgccg tcgtccgcga cccacacctt gccgatgtcg agcccgacgc
     1921 gcgtgaggaa gagttcttgc agctcggtga cccgctcgat gtggcggtcc gggtcgacgg
     1981 tgtggcgcgt ggcggggtag tcggcgaacg cggcggcgag ggtgcgtacg gcccggggga
     2041 cgtcgtcgcg ggtggcgagg cgcaccgtgg gcttgtactc ggtcatggtg ggctagctca
     2101 cgacacctga aatggaagaa aaaaactttg aaccactgtc tgaggcttga gaatgaacca
     2161 agatccaaac tcaaaaaggg caaattccaa ggagaattac atcaagtgcc aagctggcct
     2221 aacttcagtc tccacccact cagtgtgggg aaactccatc gcataaaacc cctcccccca
     2281 acctaaagac gacgtactcc aaaagcgcga gaactaatcg aggtgcctgg acggcgcccg
     2341 gtactccgtg gagtcacatg aagcgacggc tgaggacgga aaggcccttt tcctttgtgt
     2401 gggtgactca cccgcccgct ctcccgagcg ccgcgtcctc cattttgagc tccctgcagc
     2461 agggccggga agcggccatc tttccgctca cgcaactggt gccgaccggg ccagccttgc
     2521 cgcccagggc ggggcgatac acggcggcgc gaggccaggc accagagcag gccggccagc
     2581 ttgagactac ccccgtccga ttctcggtgg ccgcgctcgc aggccccgcc tcgccgaaca
     2641 tgtgcgctgg gacgcacggg ccccgtcgcc gcccgcggcc ccaaaaaccg aaataccagt
     2701 gtgcagatct tggcccgcat ttacaagact atcttgccag aaaaaaagcg tcgcagcagg
     2761 tcatcaaaaa ttttaaatgg ctagagactt atcgaaagca gcgagacagg cgcgaaggtg
     2821 ccaccagatt cgcacgcggc ggccccagcg cccaggccag gcctcaactc aagcacgagg
     2881 cgaaggggct ccttaagcgc aaggcctcga actctcccac ccacttccaa cccgaagctc
     2941 gggatcaaga atcacgtact gcagccaggt ggaagtaatt caaggcacgc aagggccata
     3001 acccgtaaag aggccaggcc cgcgggaacc acacacggca cttacctgtg ttctggcggc
     3061 aaacccgttg cgaaaaagaa cgttcacggc gactactgca cttatatacg gttctccccc
     3121 accctcggga aaaaggcgga gccagtacac gacatcactt tcccagttta ccccgcgcca
     3181 ccttctctag gcaccggttc aattgccgac ccctcccccc aacttctcgg ggactgtggg
     3241 cgatgtgcgc tctgcccact gacgggcaca tgcatggcgg taatacggtt atccacgcgg
     3301 ccgcctaatg gatcctagta ctcgagaaaa aaagcaccga ctcggtgcca ctttttcaag
     3361 ttgataacgg actagcctta tttaaacttg ctatgctgtt tccagcttag ctcttaaact
     3421 acgacaagct cgagggctcc aacaaggtgg ttctccaagg gatacttata gtctcaaaac
     3481 acacaattac tttacagtta gggtgagttt ccttttgtgc tgtttataac ttcgtataat
     3541 gtatgctata cgaagttata gaaatccaag cctatcatgt aaaatgtagc tagtattaaa
     3601 aagaacagat tatctgtctt ttatcgcaca ttaagcctct atagttacta ggaaatatta
     3661 tatgcaaatt aaccggggca ggggagtagc cgagcttctc ccacaagtcc gtgcgagggg
     3721 gccggcgcgg gcctagagat ggcgcgtcgg atctctagag cgggcccggt actaaccaaa
     3781 ctggatctct gctgtccctg taataaaccc gaaaattttg aatttttgta atttgttttt
     3841 gtaattcttt agtttgtatg tctgttgcta ttatgtctac tattctttcc cctgcactgt
     3901 accccccaat cccccctttt cttttaaaat tgtggatgaa tactgccatt tgtctgcaga
     3961 attggcgcac gcagtgccga tccgttcact aatcgaatgg atctgtctct gtctctctct
     4021 ccaccttctt cttctattcc ttcgggcctg tcgggtcccc tcggggttgg gaggtgggtc
     4081 tgaaacgata atggtgaata tccctgccta actctattca ctatagaaag tacagcaaaa
     4141 actattctta aacctaccaa gcctcctact atcattatga ataattttat ataccacagc
     4201 caatttgtta tgttaaacca attccacaaa cttgcccatt tatctaattc caataattct
     4261 tgttcattct tttcttgctg gttttgcgat tcttcaatta aggagtgtat taagcttgtg
     4321 taattgttaa tttctctgtc ccactccatc caggtcgtgt gattccaaat ctgttccaga
     4381 gatttattac tccaactagc attccaaggc acagcagtgg tgcaaatgag ttttccagag
     4441 caaccccaaa tccccaggag ctgttgatcc tttaggtatc tttccacagc caggattctt
     4501 gcctggagct gcttgatgcc ccagactgtg agttgcaaca gatgctgttg cgcctcaata
     4561 gccctcagca aattgttctg ctgctgcact ataccagaca ataattgtct ggcctgtacc
     4621 gtcagcgtca ttgacgctgc gcccatagtg cttcctgctg ctcccaagaa cccaaggaac
     4681 aaagctccta ttcccactgc tcttttttct ctctgcacca ctcttctctt tgccttggtg
     4741 ggtgctactc ctaatggttc aatttttact actttatatt tatataattc acttctccaa
     4801 ttgtccctca tatctcctcc tccaggtctg aagatcagcg gccggccgct tgctgtgcgg
     4861 tggtcttact tttgttttgc tcttcctcta tcttgtctaa agcttccttg gtgtctttta
     4921 tctctatcct ttgatgcaca caatagaggg ttgctactgt attatataat gatctaagtt
     4981 cttctgatcc tgtctgaagg gatggttgta gctgtcccag tatttgtcta cagccttctg
     5041 atgtttctaa caggccagga ttaactgcga atcgttctag ctccctgctt gcccatacta
     5101 tatgttttaa tttatatttt ttctttcccc ctggccttaa ccgaattttt tcccatcgcg
     5161 atctaattct cccccgctta atactgacgc tctcgcaccc atctctctcc ttctagcctc
     5221 cgctagtcaa aatttttggc gtactcacca gtcgccgccc ctcgcctctt gccgtgcgcg
     5281 cttcagcaag ccgagtcctg cgtcgagaga gctcctctgg tttccctttc gctttcaagt
     5341 ccctgttcgg gcgccactgc tagagatttt ccacactgac taaaagggtc tgagggatct
     5401 ctagttacca gagtcacaca acagacgggc acacactact tgaagcactc aaggcaagct
     5461 ttattgaggc ttaagcagtg ggttccctag ttagccagag agctcccagg ctcagatctg
     5521 gtctaaccag agagacccag tacaggcaaa acgcgctgct tatatagacc tcccaccgta
     5581 cacgcctacc gcccatttgc gtcaatgggg cggagttgtt acgacatttt ggaaagtccc
     5641 gttgattttg gtgccaaaac aaactcccat tgacgtcaat ggggtggaga cttggaaatc
     5701 cccgtgagtc aaaccgctat ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg
     5761 taatagcgat gactaatacg tagatgtact gccaagtagg aaagtcccat aaggtcatgt
     5821 actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg ggcgtacttg
     5881 gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac tccacccatt
     5941 gacgtcaatg gaaagtccct attggcgtta ctatgggaac atacgtcatt attgacgtca
     6001 atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt tatgtaacgc
     6061 ggaactccat atatgggcta tgaactaatg accccgtaat tgattactat taataactag
     6121 tcaataatca atgtcaacgc gtatatctgg cccgtacatc gcgaagcagc gcaaaacgcc
     6181 taaccctaag cagattcttc atgcaattgt cggtcaagcc ttgccttgtt gtagcttaaa
     6241 ttttgctcgc gcactactca gcgacctcca acacacaagc agggagcaga tactggctta
     6301 actatgcggc atcagagcag attgtactga gagtgcacca taggggatcg ggagatctcc
     6361 cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     6421 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     6481 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     6541 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     6601 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     6661 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     6721 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     6781 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     6841 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     6901 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6961 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     7021 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     7081 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     7141 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     7201 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     7261 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     7321 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     7381 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     7441 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     7501 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     7561 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     7621 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     7681 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     7741 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     7801 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     7861 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7921 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7981 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     8041 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     8101 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     8161 cttttgctgg ccttttgctc acatgctgct agagattttc cacactgact aaaagggtct
     8221 gagggatctc tagttaccag agtcacacaa cagacgggca cacactactt gaagcactca
     8281 aggcaagctt tattgaggct taagcagtgg gttccctagt tagccagaga gctcccaggc
     8341 tcagatctgg tctaaccaga gagacccagt acagtccgga tgcagctctc gggccatgtg
     8401 atgaaatgct aggcggctgt caaacctcca ctctaatact tctctctccg ggtcatccat
     8461 cccatgcagg ctcacagggt gtaacaagcg ggtgttctct ccttcattgg cttcttctac
     8521 cttctcttgc tcaactggta ctagcttgta gcaccatcca aaggtcagtg gatatctgat
     8581 ccctggccct ggtgtgtagt tctgccaatc agggaagtag ccttgtgtgt ggtagatcca
     8641 cagatcaagg atatcttgtc ttcgttggga gtgaattagc ccttccagtc cccccttttc
     8701 ttttaaaaag tggctaagat ctacagctgc cttgtaagtc attggtctta aaggtacctg
     8761 aggtgtgact ggaaaaccca cctcctcctc ctcttgtgct tctagccagg cacaatcagc
     8821 attggtagct gctgtattgc tacttgtgat tgctccatgt ttttctaggt ctcgatc
//
