LOCUS       sequence-183178-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human ECT2.
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: ECT2 B2.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..44
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    161..182
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(197..202,203..220,221..227)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    235..251
                     /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     240..262
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     259..275
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     259..275
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     294..314
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        296..314
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        342..568
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             569..749
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    796..921
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1414..1647
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1832..1876
                     /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        2054..2302
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2054..2074
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2225..2244
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2458..2575
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2644..2855
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2881..3480
                     /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(2881..2900)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3377..3397
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3496..4084
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3549..3569)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3967..3978)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4156..4389
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4467..4588
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4504..4523)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4558..4577
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4628..4763
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4783..4802)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4784..4802)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4812..4829)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4812..4828)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4821..4843)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4970..5425
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5057..5076)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5267..5288
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5451..5555
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5556..5624,5625..6416)
                     /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(5774..5793)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6587..7175
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7076..7095
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
       61 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
      121 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
      181 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
      241 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
      301 accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact
      361 cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa
      421 gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca
      481 acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt
      541 atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg
      601 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
      661 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
      721 cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa
      781 gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga
      841 ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag
      901 agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa
      961 attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc
     1021 agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga
     1081 caaatactgg gacagctaca accatccctt cagacaggat cagaagaact tagatcatta
     1141 tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag
     1201 gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg
     1261 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata
     1321 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag
     1381 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg
     1441 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt
     1501 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca
     1561 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga
     1621 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg
     1681 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa
     1741 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc
     1801 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga
     1861 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt
     1921 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat
     1981 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc
     2041 gaggggaccc agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     2101 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     2161 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     2221 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     2281 cttgtggaaa ggacgaaaca ccgtacaacg ggcgacatga aaagtttaag agctatgctg
     2341 gaaacagcat agcaagttta aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     2401 agtcggtgct ttttttgaat tctagatctt gagacaaatg gcagtattca tccacaattt
     2461 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     2521 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     2581 ttattacagg gacagcagag atccactttg gcgccggctc gagtggctcc ggtgcccgtc
     2641 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
     2701 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     2761 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     2821 ttctttttcg caacgggttt gccgccagaa cacaggtgtc gtgacgcggg atccgccacc
     2881 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     2941 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     3001 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     3061 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     3121 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     3181 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     3241 cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc
     3301 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     3361 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     3421 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     3481 acgcgttaag tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     3541 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3601 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     3661 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     3721 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     3781 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     3841 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     3901 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     3961 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     4021 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     4081 ccgcgtcgac tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     4141 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     4201 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     4261 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     4321 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     4381 tctctagcag tacgtatagt agttcatgtc atcttattat tcagtattta taacttgcaa
     4441 agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat
     4501 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4561 gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac
     4621 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4681 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     4741 gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat
     4801 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     4861 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     4921 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     4981 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     5041 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     5101 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     5161 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     5221 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5281 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     5341 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5401 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5461 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5521 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5581 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5641 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5701 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5761 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5821 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5881 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5941 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6001 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6061 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6121 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6181 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6241 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6301 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6361 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6421 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6481 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6541 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6601 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6661 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6721 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6781 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6841 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6901 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6961 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     7021 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7081 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7141 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7201 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     7261 ctgtggataa ccgtattacc gcctttgagt gagctgatac cg
//
