LOCUS       sequence-183462-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human ANAPC1.
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: ANAPC1 A2.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..38
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    155..176
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(191..196,197..214,215..221)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    229..245
                     /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     234..256
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     253..269
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     253..269
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     288..308
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        290..308
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        336..562
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             563..743
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    790..915
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1408..1641
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1826..1870
                     /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        2048..2296
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2048..2068
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2219..2238
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2451..2568
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2637..2848
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2874..3473
                     /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(2874..2893)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3370..3390
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3489..4077
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3542..3562)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3960..3971)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4149..4382
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4460..4581
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4497..4516)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4551..4570
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4621..4756
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4776..4795)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4777..4795)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4805..4822)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4805..4821)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4814..4836)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4963..5418
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5050..5069)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5260..5281
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5444..5548
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5549..5617,5618..6409)
                     /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(5767..5786)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6580..7168
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7069..7088
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
       61 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
      121 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
      181 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      241 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
      301 actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa tactcttgta
      361 gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg
      421 tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac
      481 gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa
      541 gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag cctgggagct
      601 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
      661 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
      721 gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa
      781 ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag
      841 gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat
      901 gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg
      961 ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag
     1021 ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata
     1081 ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat
     1141 acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct
     1201 ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct
     1261 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata
     1321 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
     1381 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc
     1441 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc
     1501 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt
     1561 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata
     1621 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
     1681 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac
     1741 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat
     1801 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg
     1861 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat
     1921 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa
     1981 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg
     2041 acccagagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     2101 tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     2161 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     2221 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     2281 gaaaggacga aacaccgaca acgatgattg cagcaagttt aagagctatg ctggaaacag
     2341 catagcaagt ttaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     2401 gctttttttg aattctagat cttgagacaa atggcagtat tcatccacaa ttttaaaaga
     2461 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
     2521 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
     2581 agggacagca gagatccact ttggcgccgg ctcgagtggc tccggtgccc gtcagtgggc
     2641 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg
     2701 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     2761 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     2821 tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc gggatccgcc accatgaccg
     2881 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     2941 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     3001 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     3061 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     3121 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     3181 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     3241 ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     3301 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     3361 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     3421 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt
     3481 aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3541 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3601 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3661 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3721 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3781 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3841 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     3901 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3961 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     4021 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc
     4081 gactttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa
     4141 ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac
     4201 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     4261 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     4321 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     4381 cagtacgtat agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg
     4441 aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa
     4501 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4561 caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc
     4621 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4681 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     4741 ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc
     4801 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     4861 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     4921 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     4981 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     5041 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     5101 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5161 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5221 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5281 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     5341 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5401 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5461 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5521 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5581 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5641 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5701 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5761 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5821 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5881 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5941 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6001 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6061 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6121 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6181 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6241 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6301 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6361 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6421 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6481 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6541 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6601 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6661 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6721 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6781 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6841 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6901 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6961 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7021 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7081 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7141 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7201 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     7261 taaccgtatt accgcctttg agtgagctga taccgctcgc c
//
