LOCUS       sequence-218078-        7300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human MCM6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7300)
  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 7300)
  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: MCM6 E1.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    49..70
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(85..90,91..108,109..115)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    123..139
                     /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     128..150
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     147..163
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     147..163
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     182..202
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        184..202
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        230..456
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             457..637
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    684..809
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1302..1535
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1720..1764
                     /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        1942..2190
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1942..1962
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2113..2132
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2344..2461
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2530..2741
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2767..3366
                     /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(2767..2786)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3263..3283
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3382..3970
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3435..3455)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3853..3864)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4042..4275
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4353..4474
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4390..4409)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4444..4463
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4514..4649
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4669..4688)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4670..4688)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4698..4715)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4698..4714)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4707..4729)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4856..5311
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4943..4962)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5153..5174
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5337..5441
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5442..5510,5511..6302)
                     /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(5660..5679)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6473..7061
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6962..6981
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7215..7232
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
       61 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      121 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
      181 cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat
      241 gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga
      301 gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta
      361 ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga
      421 gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc
      481 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      541 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      601 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      661 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      721 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      781 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      841 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      901 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      961 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
     1021 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
     1081 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
     1141 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     1201 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1261 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1321 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1381 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1441 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1501 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1561 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1621 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1681 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1741 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1801 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1861 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1921 ccaaccccga ggggacccag agagggccta tttcccatga ttccttcata tttgcatata
     1981 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
     2041 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     2101 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     2161 ttatatatct tgtggaaagg acgaaacacc gcacctggag gtccgcgacg gttaagagct
     2221 atgctggaaa cagcatagca agtttaaata aggctagtcc gttatcaact tgaaaaagtg
     2281 gcaccgagtc ggtgcttttt ttgaattcta gatcttgaga caaatggcag tattcatcca
     2341 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     2401 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     2461 tcgggtttat tacagggaca gcagagatcc actttggcgc cggctcgagt ggctccggtg
     2521 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
     2581 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
     2641 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
     2701 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtgtcgtga cgcgggatcc
     2761 gccaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     2821 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     2881 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     2941 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     3001 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     3061 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     3121 aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt
     3181 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     3241 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     3301 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     3361 gcctgaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga aagattgact
     3421 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3481 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     3541 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     3601 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     3661 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     3721 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     3781 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     3841 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     3901 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     3961 gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat cttagccact
     4021 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc
     4081 tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
     4141 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     4201 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     4261 ggaaaatctc tagcagtacg tatagtagtt catgtcatct tattattcag tatttataac
     4321 ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt
     4381 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4441 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc
     4501 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4561 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     4621 gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag
     4681 tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     4741 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     4801 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     4861 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4921 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4981 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     5041 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     5101 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     5161 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     5221 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5281 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     5341 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5401 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5461 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5521 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5581 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5641 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5701 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5761 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5821 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5881 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5941 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6001 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6061 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6121 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6181 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6241 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6301 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6361 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6421 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6481 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6541 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6601 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6661 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6721 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6781 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6841 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6901 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6961 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7021 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7081 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     7141 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     7201 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     7261 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
//
