LOCUS       sequence-183359-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human MISP.
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: MISP H10.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /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    2459..2576
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2645..2856
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2882..3481
                     /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(2882..2901)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3378..3398
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3497..4085
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3550..3570)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3968..3979)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4157..4390
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4468..4589
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4505..4524)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4559..4578
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4629..4764
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4784..4803)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4785..4803)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4813..4830)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4813..4829)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4822..4844)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4971..5426
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5058..5077)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5268..5289
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5452..5556
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5557..5625,5626..6417)
                     /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(5775..5794)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6588..7176
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7077..7096
                     /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 caccggatgg ggtatctggt cacggtttaa gagctatgct
     2341 ggaaacagca tagcaagttt aaataaggct agtccgttat caacttgaaa aagtggcacc
     2401 gagtcggtgc tttttttgaa ttctagatct tgagacaaat ggcagtattc atccacaatt
     2461 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
     2521 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
     2581 tttattacag ggacagcaga gatccacttt ggcgccggct cgagtggctc cggtgcccgt
     2641 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
     2701 tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
     2761 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
     2821 gttctttttc gcaacgggtt tgccgccaga acacaggtgt cgtgacgcgg gatccgccac
     2881 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     2941 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     3001 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     3061 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     3121 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     3181 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     3241 gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg
     3301 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     3361 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     3421 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     3481 aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     3541 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     3601 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     3661 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     3721 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     3781 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     3841 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     3901 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     3961 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     4021 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     4081 cccgcgtcga ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     4141 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt
     4201 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     4261 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     4321 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     4381 atctctagca gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca
     4441 aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa
     4501 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4561 ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa
     4621 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     4681 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     4741 agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta
     4801 ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     4861 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     4921 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
     4981 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     5041 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5101 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5161 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     5221 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5281 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     5341 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5401 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     5461 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5521 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5581 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5641 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5701 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5761 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5821 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5881 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5941 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6001 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6061 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6121 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6181 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6241 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6301 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6361 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6421 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6481 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6541 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6601 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6661 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6721 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6781 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6841 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6901 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6961 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7021 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7081 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7141 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7201 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     7261 tctgtggata accgtattac cgcctttgag tgagctgata c
//
