LOCUS       sequence-183121-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human TPX2.
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: TPX2 H1.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    80..101
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(116..121,122..139,140..146)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    154..170
                     /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     159..181
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     178..194
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     178..194
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     213..233
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        215..233
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        261..487
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             488..668
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    715..840
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1333..1566
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1751..1795
                     /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        1973..2221
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1973..1993
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2144..2163
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2376..2493
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2562..2773
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2799..3398
                     /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(2799..2818)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3295..3315
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3414..4002
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3467..3487)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3885..3896)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4074..4307
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4385..4506
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4422..4441)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4476..4495
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4546..4681
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4701..4720)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4702..4720)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4730..4747)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4730..4746)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4739..4761)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4888..5343
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4975..4994)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5185..5206
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5369..5473
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5474..5542,5543..6334)
                     /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(5692..5711)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6505..7093
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6994..7013
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7247..7264
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
       61 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
      121 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
      181 gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa
      241 agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt gcaacatggt
      301 aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt
      361 ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat
      421 tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata
      481 cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
      541 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
      601 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
      661 ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct
      721 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg
      781 agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc
      841 agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga
      901 aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca
      961 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa
     1021 ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc
     1081 tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag
     1141 gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg
     1201 aggaggagat atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat
     1261 tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag
     1321 agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg
     1381 cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca
     1441 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg
     1501 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca
     1561 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa
     1621 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg
     1681 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa
     1741 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa
     1801 ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg
     1861 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg
     1921 atattcacca ttatcgtttc agacccacct cccaaccccg aggggaccca gagagggcct
     1981 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattag
     2041 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     2101 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     2161 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
     2221 cgaaatccga gggggcatca tgtttaagag ctatgctgga aacagcatag caagtttaaa
     2281 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttgaattc
     2341 tagatcttga gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
     2401 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     2461 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
     2521 ccactttggc gccggctcga gtggctccgg tgcccgtcag tgggcagagc gcacatcgcc
     2581 cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg
     2641 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg
     2701 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc
     2761 cgccagaaca caggtgtcgt gacgcgggat ccgccaccat gaccgagtac aagcccacgg
     2821 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     2881 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     2941 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     3001 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     3061 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     3121 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     3181 tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     3241 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     3301 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     3361 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac
     3421 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     3481 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     3541 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     3601 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     3661 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     3721 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     3781 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg
     3841 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     3901 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     3961 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat
     4021 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
     4081 gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt
     4141 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     4201 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     4261 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta cgtatagtag
     4321 ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga
     4381 gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4441 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4501 atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact
     4561 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4621 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4681 ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     4741 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     4801 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     4861 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     4921 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     4981 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     5041 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     5101 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5161 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     5221 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     5281 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     5341 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5401 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5461 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5521 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5581 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5641 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5701 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5761 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5821 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5881 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5941 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6001 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6061 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6121 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6181 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6241 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6301 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6361 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6421 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6481 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     6541 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     6601 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     6661 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     6721 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     6781 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6841 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6901 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6961 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     7021 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     7081 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     7141 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     7201 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     7261 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc c
//
