LOCUS       sequence-183143-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human ZWILCH.
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: ZWILCH B1.6 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..46
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    163..184
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(199..204,205..222,223..229)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    237..253
                     /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     242..264
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     261..277
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     261..277
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     296..316
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        298..316
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        344..570
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             571..751
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    798..923
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1416..1649
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1834..1878
                     /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        2056..2304
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2056..2076
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2227..2246
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2460..2577
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2646..2857
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2883..3482
                     /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(2883..2902)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3379..3399
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3498..4086
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3551..3571)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3969..3980)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4158..4391
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4469..4590
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4506..4525)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4560..4579
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4630..4765
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4785..4804)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4786..4804)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4814..4831)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4814..4830)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4823..4845)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4972..5427
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5059..5078)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5269..5290
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5453..5557
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5558..5626,5627..6418)
                     /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(5776..5795)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6589..7177
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7078..7097
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
       61 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
      121 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
      181 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
      241 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat
      301 taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc ttatgcaata
      361 ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca aggagagaaa
      421 aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt attaggaagg
      481 caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg cagagatatt
      541 gtatttaagt gcctagctcg atacataaac gggtctctct ggttagacca gatctgagcc
      601 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
      661 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
      721 cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga
      781 aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa
      841 gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg
      901 agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa
      961 aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa
     1021 gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta
     1081 gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat
     1141 tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca
     1201 aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag
     1261 cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta
     1321 tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga
     1381 agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg
     1441 ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa
     1501 ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag
     1561 catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg
     1621 gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt
     1681 tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg
     1741 aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac
     1801 tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta
     1861 gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa
     1921 ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct
     1981 atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc
     2041 ccgaggggac ccagagaggg cctatttccc atgattcctt catatttgca tatacgatac
     2101 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa
     2161 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     2221 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     2281 atcttgtgga aaggacgaaa caccgcctag ccaacaagta ccttcgttta agagctatgc
     2341 tggaaacagc atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac
     2401 cgagtcggtg ctttttttga attctagatc ttgagacaaa tggcagtatt catccacaat
     2461 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata
     2521 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg
     2581 gtttattaca gggacagcag agatccactt tggcgccggc tcgagtggct ccggtgcccg
     2641 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
     2701 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
     2761 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
     2821 cgttcttttt cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg ggatccgcca
     2881 ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg
     2941 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg
     3001 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     3061 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     3121 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     3181 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     3241 agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg
     3301 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     3361 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     3421 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct
     3481 gaacgcgtta agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     3541 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     3601 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     3661 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     3721 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     3781 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3841 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     3901 cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     3961 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     4021 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     4081 ccccgcgtcg actttaagac caatgactta caaggcagct gtagatctta gccacttttt
     4141 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt
     4201 tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     4261 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     4321 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     4381 aatctctagc agtacgtata gtagttcatg tcatcttatt attcagtatt tataacttgc
     4441 aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa
     4501 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4561 tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta
     4621 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4681 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     4741 tagtgaggag gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt
     4801 attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     4861 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     4921 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct
     4981 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5041 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5101 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5161 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     5221 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5281 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     5341 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5401 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     5461 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5521 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     5581 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     5641 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     5701 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     5761 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     5821 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     5881 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     5941 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6001 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6061 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6121 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6181 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6241 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     6301 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     6361 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     6421 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     6481 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6541 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     6601 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     6661 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     6721 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     6781 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     6841 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     6901 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     6961 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7021 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7081 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7141 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7201 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     7261 ttctgtggat aaccgtatta ccgcctttga gtgagctgat ac
//
