LOCUS       sequence-182972-        7301 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 7301)
  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 7301)
  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 F5.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    74..95
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(110..115,116..133,134..140)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    148..164
                     /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     153..175
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     172..188
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     172..188
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     207..227
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        209..227
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        255..481
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             482..662
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    709..834
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1327..1560
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1745..1789
                     /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        1967..2215
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1967..1987
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2138..2157
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2370..2487
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2556..2767
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2793..3392
                     /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(2793..2812)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3289..3309
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3408..3996
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3461..3481)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3879..3890)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4068..4301
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4379..4500
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4416..4435)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4470..4489
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4540..4675
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4695..4714)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4696..4714)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4724..4741)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4724..4740)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4733..4755)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4882..5337
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4969..4988)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5179..5200
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5363..5467
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5468..5536,5537..6328)
                     /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(5686..5705)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6499..7087
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6988..7007
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7241..7258
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
       61 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
      121 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
      181 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
      241 agctgcaagc ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat
      301 gagttagcaa catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt
      361 aaggtggtac gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg
      421 aaccactgaa ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa
      481 cgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
      541 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
      601 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
      661 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
      721 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
      781 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
      841 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
      901 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
      961 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
     1021 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     1081 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     1141 caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg
     1201 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     1261 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     1321 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     1381 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     1441 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     1501 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     1561 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     1621 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     1681 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     1741 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     1801 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     1861 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     1921 accattatcg tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc
     1981 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa
     2041 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     2101 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     2161 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgatca
     2221 ttgcctcgcc gacgcgttta agagctatgc tggaaacagc atagcaagtt taaataaggc
     2281 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttga attctagatc
     2341 ttgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac
     2401 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa
     2461 aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt
     2521 tggcgccggc tcgagtggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt
     2581 ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg
     2641 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
     2701 accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag
     2761 aacacaggtg tcgtgacgcg ggatccgcca ccatgaccga gtacaagccc acggtgcgcc
     2821 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     2881 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     2941 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     3001 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     3061 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     3121 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag
     3181 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     3241 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     3301 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     3361 cctggtgcat gacccgcaag cccggtgcct gaacgcgtta agtcgacaat caacctctgg
     3421 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     3481 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     3541 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     3601 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     3661 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     3721 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     3781 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
     3841 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     3901 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     3961 agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta
     4021 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     4081 tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca
     4141 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     4201 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     4261 atccctcaga cccttttagt cagtgtggaa aatctctagc agtacgtata gtagttcatg
     4321 tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa
     4381 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4441 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4501 tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc
     4561 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4621 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg
     4681 acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac
     4741 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     4801 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     4861 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4921 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4981 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     5041 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     5101 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5161 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     5221 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     5281 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     5341 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5401 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5461 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5521 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5581 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5641 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5701 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5761 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5821 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5881 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5941 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     6001 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     6061 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     6121 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     6181 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     6241 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6301 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6361 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6421 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6481 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6541 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6601 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6661 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6721 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6781 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6841 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6901 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6961 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     7021 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     7081 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     7141 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     7201 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     7261 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg c
//
