LOCUS       sequence-273593-        8877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CENPB CRISPRi guide RNA 7.
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-g7
FEATURES             Location/Qualifiers
     source          1..8877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    183..300
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(553..586)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     590..610
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        855..1066
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1067..2005
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1962..1982
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2026..2622
                     /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(2026..2045)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2522..2542
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2632..2685
                     /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             2698..3396
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(3416..3449)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3505..4093
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3558..3578)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3976..3987)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4096..4112)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4097..4113)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4622..4802
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4864..5452)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4944..4963)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5623..6414,6415..6483))
                     /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     6246..6265
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6484..6588)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6663..6682)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6854..7233
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7235..7433
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7388..7408
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             7451..7631
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7678..7803
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8300..8533
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8718..8762
                     /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 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg
       61 acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat
      121 tagtgaacgg atcggcactg cgtgcgccaa ttctgcagac aaatggcagt attcatccac
      181 aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
      241 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
      301 cgggtttatt acagggacag cagagatcca gtttggttag taccgggccc gctctagaga
      361 tccgacgcgc catctctagg cccgcgccgg ccccctcgca cggacttgtg ggagaagctc
      421 ggctactccc ctgccccggt taatttgcat ataatatttc ctagtaacta tagaggctta
      481 atgtgcgata aaagacagat aatctgttct ttttaatact agctacattt tacatgatag
      541 gcttggattt ctataacttc gtatagcata cattatacga agttataaac agcacaaaag
      601 gaaactcacc ctaactgtaa agtaattgtg tgttttgaga ctataagtat cccttggaga
      661 accaccttgt tggttgcgct ccgacgccag gagtttaaga gctaagctgg aaacagcata
      721 gcaagtttaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
      781 tttttctcga gtactaggat ccattaggcg gccgcgtgga taaccgtatt accgccatgc
      841 atgtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
      901 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
      961 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     1021 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg
     1081 tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc
     1141 cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag
     1201 ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg cctggcctgg
     1261 gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga
     1321 taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt tctggcaaga
     1381 tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg gggccgcggg
     1441 cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg
     1501 gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct
     1561 cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc
     1621 gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg
     1681 gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc
     1741 agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt
     1801 ctcgcgcttt tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt
     1861 tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc
     1921 cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt
     1981 tcaaagtttt tttcttccat ttcaggtgtc gtgagctagc ccaccatgac cgagtacaag
     2041 cccacggtgc gcctcgccac ccgcgacgac gtcccccggg ccgtacgcac cctcgccgcc
     2101 gcgttcgccg actaccccgc cacgcgccac accgtcgacc cggaccgcca catcgagcgg
     2161 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     2221 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     2281 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     2341 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     2401 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     2461 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     2521 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     2581 gaaggaccgc gcacctggtg catgacccgc aagcccggtg ccggatcggg agagggcaga
     2641 ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaccggt cgccaccagc
     2701 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
     2761 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     2821 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
     2881 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
     2941 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     3001 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     3061 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     3121 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
     3181 aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag atccaagaaa
     3241 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
     3301 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
     3361 gacctcccta gcaaactggg gcacaagctt aattaagaat tcgtcgaggg acctaataac
     3421 ttcgtatagc atacattata cgaagttata catgtttaag ggttccggtt ccactaggta
     3481 caattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     3541 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     3601 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     3661 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     3721 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     3781 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     3841 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     3901 tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     3961 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     4021 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     4081 gccgcctccc cgcatcgata ccgtcgacct cgatcgagac ctagaaaaac atggagcaat
     4141 cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag cacaagagga
     4201 ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga cttacaaggc
     4261 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     4321 ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga
     4381 ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg gatggtgcta
     4441 caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag agaacacccg
     4501 cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag tattagagtg
     4561 gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc cggactgtac
     4621 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     4681 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     4741 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     4801 cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4861 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4921 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4981 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5041 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5101 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5161 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5221 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5281 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5341 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5401 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5461 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5521 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5581 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5641 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5701 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5761 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5821 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5881 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5941 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6001 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6061 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6121 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6181 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6241 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6301 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6361 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6421 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6481 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6541 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6601 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     6661 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     6721 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     6781 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     6841 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
     6901 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     6961 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     7021 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     7081 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     7141 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     7201 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     7261 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     7321 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     7381 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagcgcgttt
     7441 tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     7501 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     7561 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     7621 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
     7681 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
     7741 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
     7801 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
     7861 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
     7921 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
     7981 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
     8041 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
     8101 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg ctgatcttca
     8161 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
     8221 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
     8281 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
     8341 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
     8401 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
     8461 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
     8521 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
     8581 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
     8641 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
     8701 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
     8761 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag
     8821 taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagag
//
