LOCUS       sequence-183909-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human PTTG1.
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: PTTG1 A10.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    125..146
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(161..166,167..184,185..191)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    199..215
                     /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     204..226
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     223..239
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     223..239
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     258..278
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        260..278
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        306..532
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             533..713
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    760..885
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1378..1611
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1796..1840
                     /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        2018..2266
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2018..2038
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2189..2208
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2422..2539
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2608..2819
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2845..3444
                     /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(2845..2864)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3341..3361
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3460..4048
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3513..3533)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3931..3942)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4120..4353
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4431..4552
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4468..4487)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4522..4541
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4592..4727
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4747..4766)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4748..4766)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4776..4793)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4776..4792)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4785..4807)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4934..5389
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5021..5040)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5231..5252
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5415..5519
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5520..5588,5589..6380)
                     /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(5738..5757)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6551..7139
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7040..7059
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7293..7302,1..8)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
       61 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
      121 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
      181 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
      241 atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag
      301 cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
      361 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      421 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      481 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      541 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      601 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      661 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      721 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      781 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      841 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      901 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      961 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
     1021 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
     1081 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
     1141 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
     1201 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
     1261 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1321 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1381 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1441 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1501 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1561 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1621 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1681 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1741 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1801 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1861 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1921 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1981 gtttcagacc cacctcccaa ccccgagggg acccagagag ggcctatttc ccatgattcc
     2041 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
     2101 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     2161 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     2221 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgtag ctttaggtaa
     2281 ggctggtgtt taagagctat gctggaaaca gcatagcaag tttaaataag gctagtccgt
     2341 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaattctaga tcttgagaca
     2401 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     2461 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     2521 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac tttggcgccg
     2581 gctcgagtgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga
     2641 agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact
     2701 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata
     2761 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg
     2821 tgtcgtgacg cgggatccgc caccatgacc gagtacaagc ccacggtgcg cctcgccacc
     2881 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     2941 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     3001 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     3061 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     3121 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     3181 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc
     3241 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     3301 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     3361 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     3421 atgacccgca agcccggtgc ctgaacgcgt taagtcgaca atcaacctct ggattacaaa
     3481 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     3541 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     3601 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     3661 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     3721 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     3781 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     3841 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     3901 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     3961 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     4021 cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact tacaaggcag
     4081 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     4141 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
     4201 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     4261 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     4321 gaccctttta gtcagtgtgg aaaatctcta gcagtacgta tagtagttca tgtcatctta
     4381 ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta
     4441 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4501 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4561 ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4621 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     4681 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc
     4741 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     4801 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     4861 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     4921 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4981 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     5041 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     5101 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     5161 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5221 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5281 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5341 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     5401 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5461 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5521 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5581 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5641 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5701 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5761 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5821 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5881 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5941 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     6001 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     6061 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6121 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6181 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6241 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6301 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6361 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6421 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6481 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6541 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6601 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6661 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6721 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6781 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6841 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6901 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6961 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     7021 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     7081 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     7141 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     7201 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     7261 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc ag
//
