LOCUS       sequence-183425-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CBX1.
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: CBX1 A3.5 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    89..110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(125..130,131..148,149..155)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    163..179
                     /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     168..190
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     187..203
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     187..203
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     222..242
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        224..242
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        270..496
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             497..677
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    724..849
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1342..1575
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1760..1804
                     /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        1982..2230
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1982..2002
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2153..2172
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2386..2503
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2572..2783
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2809..3408
                     /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(2809..2828)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3305..3325
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3424..4012
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3477..3497)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3895..3906)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4084..4317
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4395..4516
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4432..4451)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4486..4505
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4556..4691
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4711..4730)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4712..4730)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4740..4757)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4740..4756)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4749..4771)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4898..5353
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4985..5004)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5195..5216
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5379..5483
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5484..5552,5553..6344)
                     /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(5702..5721)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6515..7103
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7004..7023
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7257..7274
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
       61 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
      121 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
      181 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
      241 gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg
      301 caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg
      361 ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct
      421 gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct
      481 agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
      541 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
      601 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
      661 gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag
      721 gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg
      781 cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc
      841 gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg
      901 ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa
      961 cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga
     1021 cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta
     1081 gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac
     1141 aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt
     1201 cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt
     1261 agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag
     1321 agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag
     1381 cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat
     1441 agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact
     1501 cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa
     1561 ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt
     1621 gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg
     1681 gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga
     1741 atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag
     1801 tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat
     1861 agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt
     1921 taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccag
     1981 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     2041 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     2101 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     2161 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     2221 acgaaacacc gaaaagttct cgaccgtcga ggtttaagag ctatgctgga aacagcatag
     2281 caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     2341 ttttgaattc tagatcttga gacaaatggc agtattcatc cacaatttta aaagaaaagg
     2401 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2461 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2521 cagcagagat ccactttggc gccggctcga gtggctccgg tgcccgtcag tgggcagagc
     2581 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct
     2641 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
     2701 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
     2761 acgggtttgc cgccagaaca caggtgtcgt gacgcgggat ccgccaccat gaccgagtac
     2821 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     2881 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     2941 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     3001 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     3061 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     3121 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     3181 ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     3241 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     3301 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     3361 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc
     3421 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     3481 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     3541 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     3601 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     3661 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     3721 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     3781 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     3841 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     3901 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     3961 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt
     4021 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     4081 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     4141 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     4201 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     4261 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     4321 cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
     4381 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4441 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4501 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc
     4561 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4621 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     4681 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     4741 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     4801 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     4861 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
     4921 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     4981 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     5041 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     5101 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     5161 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     5221 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     5281 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     5341 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5401 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5461 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5521 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5581 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5641 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     5701 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5761 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5821 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5881 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5941 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6001 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6061 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6121 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6181 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6241 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6301 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     6361 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6421 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6481 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6541 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6601 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6661 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6721 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6781 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6841 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6901 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6961 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7021 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7081 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7141 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     7201 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     7261 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa ac
//
