LOCUS       sequence-182992-        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 G9.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    77..98
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(113..118,119..136,137..143)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    151..167
                     /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     156..178
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     175..191
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     175..191
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     210..230
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        212..230
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        258..484
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             485..665
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    712..837
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1330..1563
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1748..1792
                     /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        1970..2218
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1970..1990
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2141..2160
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2374..2491
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2560..2771
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2797..3396
                     /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(2797..2816)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3293..3313
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3412..4000
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3465..3485)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3883..3894)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4072..4305
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4383..4504
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4420..4439)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4474..4493
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4544..4679
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4699..4718)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4700..4718)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4728..4745)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4728..4744)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4737..4759)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4886..5341
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4973..4992)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5183..5204
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5367..5471
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5472..5540,5541..6332)
                     /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(5690..5709)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6503..7091
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6992..7011
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7245..7262
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
       61 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
      121 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
      181 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
      241 tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac
      301 gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga
      361 agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg
      421 acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat
      481 aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
      541 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
      601 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
      661 tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga
      721 cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt
      781 acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt
      841 attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag
      901 aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt
      961 aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca
     1021 tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat
     1081 tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa
     1141 gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg
     1201 aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga
     1261 accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc
     1321 agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc
     1381 agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca
     1441 gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg
     1501 catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct
     1561 cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc
     1621 tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga
     1681 cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca
     1741 gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg
     1801 gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt
     1861 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
     1921 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccagag agggcctatt
     1981 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
     2041 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     2101 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     2161 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgt
     2221 cgaacgtttt gccaaaacgg tttaagagct atgctggaaa cagcatagca agtttaaata
     2281 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgaattcta
     2341 gatcttgaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     2401 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     2461 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     2521 actttggcgc cggctcgagt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
     2581 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
     2641 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     2701 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     2761 ccagaacaca ggtgtcgtga cgcgggatcc gccaccatga ccgagtacaa gcccacggtg
     2821 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     2881 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     2941 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     3001 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     3061 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     3121 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     3181 ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     3241 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     3301 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     3361 cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct
     3421 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     3481 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     3541 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     3601 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     3661 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     3721 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     3781 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     3841 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     3901 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     3961 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
     4021 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     4081 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
     4141 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     4201 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     4261 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt
     4321 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
     4381 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4441 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4501 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc
     4561 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4621 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     4681 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     4741 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     4801 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     4861 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4921 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     4981 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5041 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5101 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5161 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     5221 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     5281 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     5341 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5401 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5461 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5521 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5581 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5641 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5701 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5761 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5821 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5881 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5941 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6001 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6061 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6121 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6181 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6241 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6301 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6361 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6421 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6481 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6541 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6601 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6661 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6721 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6781 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6841 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6901 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6961 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7021 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7081 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7141 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     7201 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     7261 aggaagcgga agagcgccca atacgcaaac cgcctctccc cg
//
