LOCUS       sequence-182889-        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 D5.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    92..113
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(128..133,134..151,152..158)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    166..182
                     /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     171..193
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     190..206
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     190..206
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     225..245
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        227..245
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        273..499
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             500..680
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    727..852
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1345..1578
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1763..1807
                     /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        1985..2233
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1985..2005
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2156..2175
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2389..2506
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2575..2786
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2812..3411
                     /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(2812..2831)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3308..3328
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3427..4015
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3480..3500)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3898..3909)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4087..4320
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4398..4519
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4435..4454)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4489..4508
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4559..4694
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4714..4733)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4715..4733)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4743..4760)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4743..4759)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4752..4774)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4901..5356
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4988..5007)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5198..5219
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5382..5486
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5487..5555,5556..6347)
                     /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(5705..5724)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6518..7106
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7007..7026
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7260..7277
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
       61 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
      121 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
      181 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
      241 aaagggaaca aaagctggag ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc
      301 ttgcaacatg gtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc
      361 atgccgattg gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacggg
      421 tctgacatgg attggacgaa ccactgaatt gccgcattgc agagatattg tatttaagtg
      481 cctagctcga tacataaacg ggtctctctg gttagaccag atctgagcct gggagctctc
      541 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
      601 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
      661 agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca
      721 gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg
      781 cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg
      841 tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta
      901 aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta
      961 gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg
     1021 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
     1081 gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta
     1141 gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat
     1201 cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa
     1261 agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca
     1321 gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg
     1381 aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg
     1441 tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca
     1501 actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct
     1561 aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc
     1621 tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac
     1681 ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga
     1741 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
     1801 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
     1861 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
     1921 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc
     1981 cagagagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     2041 agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     2101 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     2161 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     2221 aggacgaaac accgcactac tgggaggact agctgtttaa gagctatgct ggaaacagca
     2281 tagcaagttt aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     2341 tttttttgaa ttctagatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa
     2401 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     2461 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     2521 ggacagcaga gatccacttt ggcgccggct cgagtggctc cggtgcccgt cagtgggcag
     2581 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg
     2641 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
     2701 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
     2761 gcaacgggtt tgccgccaga acacaggtgt cgtgacgcgg gatccgccac catgaccgag
     2821 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     2881 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     2941 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     3001 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     3061 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     3121 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     3181 ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     3241 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     3301 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     3361 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa
     3421 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     3481 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     3541 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     3601 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     3661 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     3721 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     3781 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     3841 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     3901 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     3961 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga
     4021 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     4081 ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg
     4141 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     4201 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     4261 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     4321 gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
     4381 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4441 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4501 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
     4561 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4621 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     4681 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
     4741 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     4801 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     4861 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     4921 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     4981 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5041 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5101 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5161 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5221 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     5281 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5341 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5401 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5461 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5521 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5581 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5641 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5701 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5761 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5821 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5881 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5941 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6001 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6061 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6121 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6181 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6241 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6301 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6361 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6421 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6481 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6541 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6601 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6661 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6721 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6781 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6841 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6901 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6961 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7021 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7081 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7141 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     7201 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     7261 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg ca
//
