LOCUS       sequence-183416-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CBX5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7302)
  AUTHORS   McKinley KL, Cheeseman IM
  TITLE     Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the
            Diversity of p53-Dependent Responses to Cell-Cycle Defects.
  JOURNAL   Dev Cell. 2017 Feb 27;40(4):405-420.e2. doi: 
            10.1016/j.devcel.2017.01.012. Epub 2017 Feb 16.
  PUBMED    28216383
REFERENCE   2  (bases 1 to 7302)
  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.devcel.2017.01.012"; journalName: "Dev Cell"; date: 
            "2017-02-27- 27"; volume: "40"; issue: "4"; pages: "405-420.e2"
COMMENT     Sequence Label: CBX5 A1.5 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    101..122
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(137..142,143..160,161..167)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    175..191
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     180..202
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     199..215
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     199..215
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     234..254
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        236..254
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        282..508
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             509..689
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    736..861
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1354..1587
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1772..1816
                     /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"
     promoter        1994..2242
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1994..2014
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2165..2184
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2398..2515
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2584..2795
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2821..3420
                     /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(2821..2840)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3317..3337
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3436..4024
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3489..3509)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3907..3918)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4096..4329
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4407..4528
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4444..4463)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4498..4517
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4568..4703
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4723..4742)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4724..4742)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4752..4769)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4752..4768)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4761..4783)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4910..5365
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4997..5016)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5207..5228
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5391..5495
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5496..5564,5565..6356)
                     /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(5714..5733)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6527..7115
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7016..7035
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7269..7286
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
       61 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
      121 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
      181 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
      241 accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact
      301 cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa
      361 gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca
      421 acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt
      481 atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg
      541 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
      601 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
      661 cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa
      721 gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga
      781 ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag
      841 agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa
      901 attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc
      961 agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga
     1021 caaatactgg gacagctaca accatccctt cagacaggat cagaagaact tagatcatta
     1081 tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag
     1141 gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg
     1201 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata
     1261 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag
     1321 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg
     1381 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt
     1441 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca
     1501 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga
     1561 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg
     1621 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa
     1681 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc
     1741 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga
     1801 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt
     1861 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat
     1921 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc
     1981 gaggggaccc agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     2041 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     2101 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     2161 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     2221 cttgtggaaa ggacgaaaca ccgctagaca ggcgcgtggt taagtttaag agctatgctg
     2281 gaaacagcat agcaagttta aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     2341 agtcggtgct ttttttgaat tctagatctt gagacaaatg gcagtattca tccacaattt
     2401 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     2461 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     2521 ttattacagg gacagcagag atccactttg gcgccggctc gagtggctcc ggtgcccgtc
     2581 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
     2641 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     2701 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     2761 ttctttttcg caacgggttt gccgccagaa cacaggtgtc gtgacgcggg atccgccacc
     2821 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     2881 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     2941 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     3001 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     3061 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     3121 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     3181 cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc
     3241 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     3301 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     3361 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     3421 acgcgttaag tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     3481 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3541 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     3601 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     3661 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     3721 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     3781 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     3841 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     3901 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     3961 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     4021 ccgcgtcgac tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     4081 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     4141 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     4201 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     4261 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     4321 tctctagcag tacgtatagt agttcatgtc atcttattat tcagtattta taacttgcaa
     4381 agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat
     4441 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4501 gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac
     4561 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4621 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     4681 gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat
     4741 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     4801 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     4861 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     4921 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4981 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     5041 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     5101 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     5161 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5221 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     5281 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5341 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5401 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5461 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5521 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5581 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5641 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5701 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5761 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5821 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5881 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5941 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6001 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6061 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6121 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6181 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6241 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6301 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6361 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6421 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6481 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6541 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6601 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6661 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6721 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6781 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6841 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6901 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6961 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7021 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7081 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7141 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     7201 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     7261 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cc
//
