LOCUS       sequence-182689-        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 D6.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    82..103
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(118..123,124..141,142..148)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    156..172
                     /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     161..183
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     180..196
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     180..196
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     215..235
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        217..235
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        263..489
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             490..670
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    717..842
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1335..1568
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1753..1797
                     /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        1975..2223
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1975..1995
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2146..2165
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2379..2496
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2565..2776
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2802..3401
                     /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(2802..2821)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3298..3318
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3417..4005
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3470..3490)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3888..3899)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4077..4310
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4388..4509
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4425..4444)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4479..4498
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4549..4684
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4704..4723)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4705..4723)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4733..4750)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4733..4749)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4742..4764)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4891..5346
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4978..4997)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5188..5209
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5372..5476
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5477..5545,5546..6337)
                     /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(5695..5714)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6508..7096
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6997..7016
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7250..7267
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
       61 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
      121 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
      181 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
      241 aaagctggag ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg
      301 gtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc atgccgattg
      361 gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacggg tctgacatgg
      421 attggacgaa ccactgaatt gccgcattgc agagatattg tatttaagtg cctagctcga
      481 tacataaacg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
      541 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
      601 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
      661 atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct
      721 ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
      781 tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg
      841 tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg
      901 gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg
      961 cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac
     1021 aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc
     1081 tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag
     1141 aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct
     1201 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     1261 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     1321 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     1381 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     1441 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     1501 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     1561 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     1621 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     1681 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     1741 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     1801 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     1861 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     1921 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cagagagggc
     1981 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     2041 agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     2101 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     2161 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     2221 accgcaagct agtcctccca gtaggtttaa gagctatgct ggaaacagca tagcaagttt
     2281 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttgaa
     2341 ttctagatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
     2401 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     2461 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
     2521 gatccacttt ggcgccggct cgagtggctc cggtgcccgt cagtgggcag agcgcacatc
     2581 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     2641 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     2701 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     2761 tgccgccaga acacaggtgt cgtgacgcgg gatccgccac catgaccgag tacaagccca
     2821 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     2881 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     2941 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     3001 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     3061 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     3121 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     3181 ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     3241 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     3301 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     3361 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc
     3421 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     3481 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     3541 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     3601 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     3661 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     3721 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     3781 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     3841 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3901 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     3961 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc
     4021 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     4081 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg
     4141 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     4201 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     4261 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag
     4321 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag
     4381 tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     4441 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     4501 tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta
     4561 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4621 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4681 ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
     4741 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     4801 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     4861 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     4921 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     4981 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5041 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5101 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5161 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5221 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     5281 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     5341 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5401 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     5461 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5521 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5581 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     5641 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     5701 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5761 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5821 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5881 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5941 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6001 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6061 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6121 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6181 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     6241 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6301 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     6361 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6421 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6481 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6541 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6601 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     6661 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6721 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6781 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6841 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6901 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6961 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7021 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7081 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7141 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     7201 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     7261 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ct
//
