LOCUS       sequence-182955-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human MCM2.
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: MCM2 F6.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /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    2417..2534
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2603..2814
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2840..3439
                     /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(2840..2859)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3336..3356
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3455..4043
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3508..3528)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3926..3937)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4115..4348
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4426..4547
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4463..4482)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4517..4536
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4587..4722
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4742..4761)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4743..4761)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4771..4788)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4771..4787)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4780..4802)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4929..5384
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5016..5035)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5226..5247
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5410..5514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5515..5583,5584..6375)
                     /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(5733..5752)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6546..7134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7035..7054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7288..7302,1..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 cgaggtgagg gcatcagtac
     2281 gcgtttaaga gctatgctgg aaacagcata gcaagtttaa ataaggctag tccgttatca
     2341 acttgaaaaa gtggcaccga gtcggtgctt tttttgaatt ctagatcttg agacaaatgg
     2401 cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa
     2461 gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa
     2521 aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg cgccggctcg
     2581 agtggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg
     2641 gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa
     2701 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
     2761 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtgtcg
     2821 tgacgcggga tccgccacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     2881 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     2941 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     3001 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     3061 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     3121 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     3181 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggagtct cgcccgacca
     3241 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     3301 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     3361 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     3421 ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa cctctggatt acaaaatttg
     3481 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     3541 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     3601 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     3661 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     3721 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     3781 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     3841 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
     3901 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     3961 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     4021 ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta
     4081 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     4141 agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg
     4201 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     4261 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     4321 ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca tcttattatt
     4381 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca
     4441 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4501 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc
     4561 tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     4621 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     4681 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc
     4741 gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg
     4801 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     4861 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     4921 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4981 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     5041 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     5101 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     5161 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5221 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5281 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5341 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     5401 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5461 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5521 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5581 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5641 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5701 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5761 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5821 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5881 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5941 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6001 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6061 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6121 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6181 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6241 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6301 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6361 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6421 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6481 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6541 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6601 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6661 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6721 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6781 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6841 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6901 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6961 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7021 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7081 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7141 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7201 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7261 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gc
//
