LOCUS       sequence-183141-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human ZW10.
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: ZW10 H10.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..33
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    150..171
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(186..191,192..209,210..216)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    224..240
                     /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     229..251
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     248..264
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     248..264
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     283..303
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        285..303
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        331..557
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             558..738
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    785..910
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1403..1636
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1821..1865
                     /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        2043..2291
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2043..2063
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2214..2233
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2447..2564
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2633..2844
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2870..3469
                     /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(2870..2889)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3366..3386
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3485..4073
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3538..3558)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3956..3967)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4145..4378
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4456..4577
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4493..4512)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4547..4566
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4617..4752
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4772..4791)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4773..4791)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4801..4818)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4801..4817)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4810..4832)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4959..5414
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5046..5065)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5256..5277
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5440..5544
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5545..5613,5614..6405)
                     /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(5763..5782)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6576..7164
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7065..7084
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
       61 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      121 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
      181 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
      241 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
      301 agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt
      361 gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat
      421 gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc
      481 tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc
      541 tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg
      601 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
      661 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
      721 tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga
      781 ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg
      841 gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg
      901 cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag
      961 gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga
     1021 acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg
     1081 acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt
     1141 agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga
     1201 caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct
     1261 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag
     1321 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
     1381 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
     1441 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta
     1501 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
     1561 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa
     1621 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
     1681 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct
     1741 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
     1801 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
     1861 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga
     1921 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag
     1981 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggaccca
     2041 gagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     2101 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     2161 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     2221 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     2281 gacgaaacac cgaagttttg gcacactccg gggtttaaga gctatgctgg aaacagcata
     2341 gcaagtttaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     2401 tttttgaatt ctagatcttg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     2461 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2521 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2581 acagcagaga tccactttgg cgccggctcg agtggctccg gtgcccgtca gtgggcagag
     2641 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc
     2701 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
     2761 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
     2821 aacgggtttg ccgccagaac acaggtgtcg tgacgcggga tccgccacca tgaccgagta
     2881 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
     2941 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
     3001 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     3061 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     3121 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     3181 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     3241 cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     3301 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     3361 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     3421 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt
     3481 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     3541 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     3601 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     3661 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     3721 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     3781 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     3841 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     3901 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     3961 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     4021 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact
     4081 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     4141 ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg
     4201 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     4261 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     4321 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     4381 acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     4441 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4501 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4561 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc
     4621 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4681 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     4741 tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     4801 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     4861 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     4921 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     4981 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     5041 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5101 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5161 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5221 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5281 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     5341 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5401 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5461 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5521 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5581 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5641 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5701 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5761 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5821 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5881 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5941 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6001 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6061 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6121 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6181 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6241 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6301 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6361 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6421 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6481 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6541 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6601 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6661 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6721 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6781 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6841 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6901 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6961 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     7021 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7081 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7141 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7201 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     7261 cgtattaccg cctttgagtg agctgatacc gctcgccgca gc
//
