LOCUS       sequence-183564-        7277 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human MCM7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7277)
  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 7277)
  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: MCM7 E3.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7277
                     /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..2182
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     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    2321..2438
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2507..2718
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2744..3343
                     /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(2744..2763)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3240..3260
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3359..3947
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3412..3432)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3830..3841)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4019..4252
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4330..4451
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4367..4386)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4421..4440
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4491..4626
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4646..4665)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4647..4665)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4675..4692)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4675..4691)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4684..4706)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4833..5288
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4920..4939)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5130..5151
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5314..5418
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5419..5487,5488..6279)
                     /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(5637..5656)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6450..7038
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6939..6958
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7192..7209
                     /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 acgaaagttt aagagctatg ctggaaacag catagcaagt
     2221 ttaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg
     2281 aattctagat cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
     2341 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
     2401 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
     2461 gagatccact ttggcgccgg ctcgagtggc tccggtgccc gtcagtgggc agagcgcaca
     2521 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga
     2581 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     2641 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     2701 tttgccgcca gaacacaggt gtcgtgacgc gggatccgcc accatgaccg agtacaagcc
     2761 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     2821 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     2881 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     2941 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     3001 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     3061 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     3121 caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     3181 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     3241 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     3301 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa
     3361 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3421 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3481 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     3541 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     3601 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     3661 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     3721 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     3781 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     3841 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     3901 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga
     3961 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     4021 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     4081 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     4141 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     4201 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat
     4261 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
     4321 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4381 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4441 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc
     4501 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4561 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4621 gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     4681 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     4741 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     4801 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     4861 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     4921 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4981 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5041 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5101 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5161 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     5221 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     5281 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5341 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5401 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5461 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5521 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5581 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5641 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5701 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5761 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5821 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5881 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5941 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6001 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6061 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6121 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6181 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6241 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6301 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6361 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6421 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6481 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6541 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6601 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6661 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6721 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6781 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6841 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6901 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6961 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7021 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7081 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     7141 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     7201 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     7261 attcattaat gcagctg
//
