LOCUS       sequence-183223-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human TBCB.
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: TBCB E11.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    121..142
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(157..162,163..180,181..187)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    195..211
                     /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     200..222
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     219..235
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     219..235
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     254..274
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        256..274
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        302..528
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             529..709
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    756..881
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1374..1607
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1792..1836
                     /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        2014..2262
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2014..2034
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2185..2204
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2418..2535
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2604..2815
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2841..3440
                     /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(2841..2860)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3337..3357
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3456..4044
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3509..3529)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3927..3938)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4116..4349
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4427..4548
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4464..4483)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4518..4537
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4588..4723
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4743..4762)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4744..4762)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4772..4789)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4772..4788)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4781..4803)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4930..5385
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5017..5036)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5227..5248
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5411..5515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5516..5584,5585..6376)
                     /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(5734..5753)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6547..7135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7036..7055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7289..7302,1..4)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
       61 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
      121 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
      181 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      241 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta
      301 atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat
      361 gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat
      421 cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg
      481 ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg
      541 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      601 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      661 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
      721 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
      781 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
      841 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
      901 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
      961 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
     1021 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
     1081 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1141 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1201 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1261 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1321 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1381 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1441 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1501 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1561 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1621 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1681 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     1741 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     1801 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     1861 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     1921 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     1981 cagacccacc tcccaacccc gaggggaccc agagagggcc tatttcccat gattccttca
     2041 tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac
     2101 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     2161 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     2221 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgcacaaga agcctacgac
     2281 caggtttaag agctatgctg gaaacagcat agcaagttta aataaggcta gtccgttatc
     2341 aacttgaaaa agtggcaccg agtcggtgct ttttttgaat tctagatctt gagacaaatg
     2401 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2461 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2521 aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc
     2581 gagtggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt
     2641 ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga
     2701 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
     2761 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc
     2821 gtgacgcggg atccgccacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     2881 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     2941 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     3001 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     3061 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     3121 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     3181 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc
     3241 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     3301 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     3361 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     3421 cccgcaagcc cggtgcctga acgcgttaag tcgacaatca acctctggat tacaaaattt
     3481 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     3541 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     3601 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     3661 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     3721 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     3781 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     3841 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
     3901 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     3961 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     4021 tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca aggcagctgt
     4081 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg
     4141 aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg
     4201 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
     4261 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
     4321 cttttagtca gtgtggaaaa tctctagcag tacgtatagt agttcatgtc atcttattat
     4381 tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc
     4441 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4501 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct
     4561 ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4621 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     4681 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt
     4741 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
     4801 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     4861 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     4921 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4981 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     5041 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     5101 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     5161 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     5221 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     5281 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     5341 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     5401 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5461 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5521 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5581 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5641 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5701 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5761 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5821 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5881 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5941 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6001 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6061 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6121 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6181 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6241 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6301 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6361 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6421 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6481 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6541 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6601 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6661 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6721 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6781 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6841 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6901 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6961 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     7021 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7081 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     7141 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     7201 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     7261 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg ag
//
