LOCUS       sequence-182998-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human MCM4.
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: MCM4 F7.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    36..57
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(72..77,78..95,96..102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    110..126
                     /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     115..137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     134..150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     134..150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     169..189
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        171..189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        217..443
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             444..624
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    671..796
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1289..1522
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1707..1751
                     /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        1929..2177
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1929..1949
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2100..2119
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2333..2450
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2519..2730
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2756..3355
                     /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(2756..2775)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3252..3272
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3371..3959
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3424..3444)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3842..3853)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4031..4264
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4342..4463
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4379..4398)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4433..4452
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4503..4638
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4658..4677)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4659..4677)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4687..4704)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4687..4703)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4696..4718)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4845..5300
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4932..4951)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5142..5163
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5326..5430
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5431..5499,5500..6291)
                     /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(5649..5668)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6462..7050
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6951..6970
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7204..7221
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
       61 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
      121 taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct
      181 cactaaaggg aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt
      241 agtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc
      301 gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga
      361 cgggtctgac atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta
      421 agtgcctagc tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc
      481 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
      541 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
      601 agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa
      661 accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga
      721 ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga
      781 tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg
      841 gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga
      901 gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat
      961 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
     1021 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
     1081 tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc
     1141 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
     1201 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
     1261 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
     1321 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
     1381 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
     1441 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
     1501 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
     1561 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
     1621 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
     1681 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat
     1741 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
     1801 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga
     1861 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
     1921 gacccagaga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     1981 ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     2041 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     2101 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     2161 ggaaaggacg aaacaccgcg aggattccac ctccacgggt ttaagagcta tgctggaaac
     2221 agcatagcaa gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     2281 gtgctttttt tgaattctag atcttgagac aaatggcagt attcatccac aattttaaaa
     2341 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     2401 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     2461 acagggacag cagagatcca ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg
     2521 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc
     2581 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
     2641 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
     2701 tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac gcgggatccg ccaccatgac
     2761 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     2821 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     2881 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     2941 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     3001 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     3061 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     3121 gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     3181 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     3241 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     3301 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg
     3361 ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     3421 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     3481 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     3541 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     3601 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     3661 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3721 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     3781 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     3841 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     3901 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg
     3961 tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     4021 aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt
     4081 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     4141 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     4201 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     4261 agcagtacgt atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     4321 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc
     4381 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4441 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc
     4501 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     4561 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     4621 gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc
     4681 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     4741 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     4801 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     4861 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4921 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4981 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5041 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5101 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5161 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     5221 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5281 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5341 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5401 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5461 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5521 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5581 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5641 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5701 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5761 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5821 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5881 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5941 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6001 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6061 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6121 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     6181 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6241 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6301 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6361 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6421 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6481 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6541 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6601 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6661 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6721 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6781 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6841 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6901 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6961 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7021 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7081 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     7141 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     7201 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     7261 gcgcgttggc cgattcatta atgcagctgg cacgacaggt tt
//
