LOCUS       sequence-183135-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human TRIP13.
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: TRIP13 H4.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..20
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    137..158
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(173..178,179..196,197..203)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    211..227
                     /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     216..238
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     235..251
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     235..251
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     270..290
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        272..290
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        318..544
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             545..725
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    772..897
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1390..1623
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1808..1852
                     /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        2030..2278
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2030..2050
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2201..2220
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2433..2550
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2619..2830
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2856..3455
                     /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(2856..2875)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3352..3372
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3471..4059
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3524..3544)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3942..3953)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4131..4364
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4442..4563
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4479..4498)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4533..4552
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4603..4738
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4758..4777)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4759..4777)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4787..4804)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4787..4803)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4796..4818)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4945..5400
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5032..5051)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5242..5263
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5426..5530
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5531..5599,5600..6391)
                     /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(5749..5768)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6562..7150
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7051..7070
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
       61 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
      121 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
      181 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
      241 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
      301 tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac
      361 gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga
      421 agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg
      481 acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat
      541 aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
      601 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
      661 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
      721 tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga
      781 cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt
      841 acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt
      901 attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag
      961 aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt
     1021 aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca
     1081 tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat
     1141 tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa
     1201 gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg
     1261 aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga
     1321 accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc
     1381 agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc
     1441 agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca
     1501 gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg
     1561 catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct
     1621 cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc
     1681 tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga
     1741 cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca
     1801 gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg
     1861 gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt
     1921 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
     1981 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccagag agggcctatt
     2041 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
     2101 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     2161 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     2221 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgt
     2281 gcatgcactc aaatcgatgt ttaagagcta tgctggaaac agcatagcaa gtttaaataa
     2341 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgaattctag
     2401 atcttgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
     2461 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     2521 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     2581 ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
     2641 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
     2701 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
     2761 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
     2821 cagaacacag gtgtcgtgac gcgggatccg ccaccatgac cgagtacaag cccacggtgc
     2881 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
     2941 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
     3001 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
     3061 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
     3121 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
     3181 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
     3241 gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
     3301 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
     3361 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
     3421 gcacctggtg catgacccgc aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc
     3481 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     3541 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     3601 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     3661 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     3721 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     3781 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     3841 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     3901 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     3961 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     4021 ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac
     4081 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     4141 aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga
     4201 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     4261 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     4321 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt atagtagttc
     4381 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
     4441 gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4501 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4561 ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg
     4621 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4681 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4741 gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     4801 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     4861 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     4921 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     4981 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     5041 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5101 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5161 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5221 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5281 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5341 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     5401 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5461 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5521 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5581 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5641 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5701 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5761 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5821 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5881 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5941 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6001 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6061 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6121 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6181 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6241 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6301 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     6361 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     6421 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6481 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6541 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6601 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6661 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6721 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6781 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6841 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6901 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6961 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     7021 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     7081 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     7141 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     7201 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     7261 tgagtgagct gataccgctc gccgcagccg aacgaccgag c
//
