LOCUS       sequence-182707-        7304 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CDC6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7304)
  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 7304)
  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: CDC6 B6.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7304
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    120..141
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(156..161,162..179,180..186)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    194..210
                     /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     199..221
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     218..234
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     218..234
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     253..273
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        255..273
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        301..527
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             528..708
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    755..880
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1373..1606
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1791..1835
                     /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        2013..2261
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2013..2033
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2184..2203
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2419..2536
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2605..2816
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2842..3441
                     /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(2842..2861)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3338..3358
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3457..4045
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3510..3530)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3928..3939)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4117..4350
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4428..4549
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4465..4484)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4519..4538
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4589..4724
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4744..4763)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4745..4763)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4773..4790)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4773..4789)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4782..4804)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4931..5386
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5018..5037)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5228..5249
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5412..5516
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5517..5585,5586..6377)
                     /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(5735..5754)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6548..7136
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7037..7056
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7290..7304,1..3)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
       61 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
      121 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
      181 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
      241 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa
      301 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
      361 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      421 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      481 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      541 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      601 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      661 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      721 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      781 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      841 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      901 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      961 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
     1021 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
     1081 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
     1141 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
     1201 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
     1261 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1321 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1381 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1441 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1501 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1561 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1621 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1681 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1741 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1801 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1861 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1921 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1981 agacccacct cccaaccccg aggggaccca gagagggcct atttcccatg attccttcat
     2041 atttgcatat acgatacaag gctgttagag agataattag aattaatttg actgtaaaca
     2101 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     2161 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     2221 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggcttttac acgaggagaa
     2281 cagggtttaa gagctatgct ggaaacagca tagcaagttt aaataaggct agtccgttat
     2341 caacttgaaa aagtggcacc gagtcggtgc tttttttgaa ttctagatct tgagacaaat
     2401 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga
     2461 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac
     2521 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt ggcgccggct
     2581 cgagtggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
     2641 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg
     2701 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     2761 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtgt
     2821 cgtgacgcgg gatccgccac catgaccgag tacaagccca cggtgcgcct cgccacccgc
     2881 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     2941 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     3001 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     3061 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     3121 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     3181 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac
     3241 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     3301 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     3361 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     3421 acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt
     3481 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     3541 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     3601 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     3661 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     3721 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     3781 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     3841 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg
     3901 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     3961 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     4021 atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg
     4081 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
     4141 gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct
     4201 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     4261 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     4321 ccttttagtc agtgtggaaa atctctagca gtacgtatag tagttcatgt catcttatta
     4381 ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg
     4441 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4501 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc
     4561 tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     4621 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     4681 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat
     4741 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac
     4801 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     4861 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     4921 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4981 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     5041 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5101 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     5161 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5221 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     5281 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5341 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     5401 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5461 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5521 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5581 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5641 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5701 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5761 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5821 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5881 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5941 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6001 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6061 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6121 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6181 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     6241 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6301 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6361 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6421 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6481 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6541 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6601 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6661 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6721 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6781 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6841 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6901 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6961 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     7021 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     7081 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     7141 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     7201 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     7261 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagc
//
