LOCUS       sequence-183018-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human NDC80.
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: NDC80 F12.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    61..82
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(97..102,103..120,121..127)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    135..151
                     /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     140..162
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     159..175
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     159..175
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     194..214
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        196..214
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        242..468
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             469..649
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    696..821
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1314..1547
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1732..1776
                     /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        1954..2202
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1954..1974
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2125..2144
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2357..2474
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2543..2754
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2780..3379
                     /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(2780..2799)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3276..3296
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3395..3983
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3448..3468)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3866..3877)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4055..4288
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4366..4487
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4403..4422)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4457..4476
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4527..4662
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4682..4701)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4683..4701)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4711..4728)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4711..4727)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4720..4742)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4869..5324
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4956..4975)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5166..5187
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5350..5454
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5455..5523,5524..6315)
                     /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(5673..5692)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6486..7074
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6975..6994
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7228..7245
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
       61 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
      121 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      181 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta
      241 atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat
      301 gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat
      361 cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg
      421 ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg
      481 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      541 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      601 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
      661 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
      721 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
      781 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
      841 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
      901 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
      961 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
     1021 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1081 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1141 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1201 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1261 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1321 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1381 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1441 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1501 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1561 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1621 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     1681 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     1741 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     1801 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     1861 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     1921 cagacccacc tcccaacccc gaggggaccc agagagggcc tatttcccat gattccttca
     1981 tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac
     2041 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     2101 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     2161 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgaaagaaa agtctcgcta
     2221 ttgtttaaga gctatgctgg aaacagcata gcaagtttaa ataaggctag tccgttatca
     2281 acttgaaaaa gtggcaccga gtcggtgctt tttttgaatt ctagatcttg agacaaatgg
     2341 cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa
     2401 gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa
     2461 aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg cgccggctcg
     2521 agtggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg
     2581 gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa
     2641 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
     2701 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtgtcg
     2761 tgacgcggga tccgccacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     2821 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     2881 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     2941 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     3001 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     3061 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     3121 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggagtct cgcccgacca
     3181 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     3241 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     3301 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     3361 ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa cctctggatt acaaaatttg
     3421 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     3481 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     3541 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     3601 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     3661 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     3721 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     3781 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
     3841 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     3901 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     3961 ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta
     4021 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     4081 agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg
     4141 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     4201 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     4261 ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca tcttattatt
     4321 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca
     4381 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4441 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc
     4501 tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     4561 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     4621 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc
     4681 gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg
     4741 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     4801 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     4861 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4921 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4981 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     5041 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     5101 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5161 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5221 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5281 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     5341 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5401 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5461 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5521 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5581 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5641 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5701 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5761 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5821 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5881 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5941 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6001 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6061 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6121 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6181 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6241 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6301 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6361 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6421 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6481 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6541 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6601 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6661 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6721 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6781 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6841 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6901 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6961 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7021 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7081 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7141 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7201 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     7261 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc a
//
