LOCUS       sequence-183366-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human SASS6.
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: SASS6 G2.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     31..48
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    165..186
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(201..206,207..224,225..231)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    239..255
                     /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     244..266
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     263..279
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     263..279
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     298..318
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        300..318
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        346..572
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             573..753
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    800..925
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1418..1651
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1836..1880
                     /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        2058..2306
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2058..2078
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2229..2248
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2462..2579
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2648..2859
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2885..3484
                     /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(2885..2904)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3381..3401
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3500..4088
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3553..3573)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3971..3982)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4160..4393
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4471..4592
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4508..4527)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4562..4581
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4632..4767
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4787..4806)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4788..4806)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4816..4833)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4816..4832)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4825..4847)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4974..5429
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5061..5080)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5271..5292
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5455..5559
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5560..5628,5629..6420)
                     /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(5778..5797)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6591..7179
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7080..7099
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
       61 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
      121 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
      181 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
      241 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
      301 attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa
      361 tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga
      421 aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa
      481 ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata
      541 ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag
      601 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
      661 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
      721 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
      781 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
      841 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
      901 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
      961 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
     1021 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
     1081 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
     1141 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
     1201 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     1261 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
     1321 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
     1381 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
     1441 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
     1501 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
     1561 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
     1621 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
     1681 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
     1741 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
     1801 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
     1861 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
     1921 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
     1981 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
     2041 ccccgagggg acccagagag ggcctatttc ccatgattcc ttcatatttg catatacgat
     2101 acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag atattagtac
     2161 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     2221 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     2281 atatcttgtg gaaaggacga aacaccgcct gcaacgggac tagttgggtt taagagctat
     2341 gctggaaaca gcatagcaag tttaaataag gctagtccgt tatcaacttg aaaaagtggc
     2401 accgagtcgg tgcttttttt gaattctaga tcttgagaca aatggcagta ttcatccaca
     2461 attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
     2521 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc
     2581 gggtttatta cagggacagc agagatccac tttggcgccg gctcgagtgg ctccggtgcc
     2641 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
     2701 aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
     2761 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
     2821 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg tgtcgtgacg cgggatccgc
     2881 caccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     2941 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     3001 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     3061 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     3121 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     3181 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     3241 ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct
     3301 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     3361 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     3421 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     3481 ctgaacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     3541 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3601 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3661 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     3721 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     3781 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     3841 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     3901 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     3961 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     4021 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     4081 ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     4141 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
     4201 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     4261 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     4321 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     4381 aaaatctcta gcagtacgta tagtagttca tgtcatctta ttattcagta tttataactt
     4441 gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac
     4501 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4561 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc
     4621 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     4681 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     4741 agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc
     4801 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     4861 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     4921 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     4981 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     5041 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     5101 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     5161 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     5221 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     5281 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     5341 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     5401 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     5461 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5521 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5581 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5641 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5701 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5761 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5821 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5881 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5941 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6001 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6061 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6121 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6181 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6241 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     6301 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6361 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6421 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6481 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6541 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6601 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6661 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6721 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6781 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6841 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6901 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6961 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7021 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7081 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7141 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7201 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     7261 gattctgtgg ataaccgtat taccgccttt gagtgagctg at
//
