LOCUS       sequence-182961-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human SKA1.
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: SKA1 H4.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    112..133
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(148..153,154..171,172..178)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    186..202
                     /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     191..213
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     210..226
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     210..226
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     245..265
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        247..265
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        293..519
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             520..700
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    747..872
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1365..1598
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1783..1827
                     /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        2005..2253
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2005..2025
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2176..2195
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2409..2526
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2595..2806
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2832..3431
                     /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(2832..2851)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3328..3348
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3447..4035
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3500..3520)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3918..3929)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4107..4340
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4418..4539
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4455..4474)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4509..4528
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4579..4714
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4734..4753)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4735..4753)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4763..4780)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4763..4779)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4772..4794)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4921..5376
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5008..5027)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5218..5239
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5402..5506
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5507..5575,5576..6367)
                     /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(5725..5744)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6538..7126
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7027..7046
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7280..7297
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
       61 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
      121 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      181 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      241 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt aatgtagtct
      301 tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca tgccttacaa
      361 ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga tcgtgcctta
      421 ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt gccgcattgc
      481 agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg gttagaccag
      541 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
      601 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
      661 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
      721 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
      781 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
      841 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
      901 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
      961 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
     1021 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
     1081 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
     1141 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
     1201 cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
     1261 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
     1321 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
     1381 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
     1441 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
     1501 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
     1561 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
     1621 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
     1681 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
     1741 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
     1801 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
     1861 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
     1921 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
     1981 ctcccaaccc cgaggggacc cagagagggc ctatttccca tgattccttc atatttgcat
     2041 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
     2101 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     2161 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     2221 gctttatata tcttgtggaa aggacgaaac accgcagttt tcaaggtagg ttccgtttaa
     2281 gagctatgct ggaaacagca tagcaagttt aaataaggct agtccgttat caacttgaaa
     2341 aagtggcacc gagtcggtgc tttttttgaa ttctagatct tgagacaaat ggcagtattc
     2401 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     2461 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     2521 aattttcggg tttattacag ggacagcaga gatccacttt ggcgccggct cgagtggctc
     2581 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     2641 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     2701 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     2761 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtgt cgtgacgcgg
     2821 gatccgccac catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     2881 ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     2941 tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     3001 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     3061 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     3121 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     3181 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac caccagggca
     3241 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     3301 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     3361 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     3421 ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat
     3481 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     3541 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     3601 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     3661 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     3721 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     3781 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     3841 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     3901 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     3961 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     4021 gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg tagatcttag
     4081 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
     4141 tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     4201 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     4261 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     4321 agtgtggaaa atctctagca gtacgtatag tagttcatgt catcttatta ttcagtattt
     4381 ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa
     4441 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4501 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc
     4561 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4621 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4681 ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata
     4741 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     4801 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     4861 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg
     4921 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4981 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5041 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5101 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     5161 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5221 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     5281 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5341 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     5401 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5461 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5521 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5581 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5641 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5701 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5761 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5821 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5881 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5941 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6001 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     6061 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6121 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6181 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     6241 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6301 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6361 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6421 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6481 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6541 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6601 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6661 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6721 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6781 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6841 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6901 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6961 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7021 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7081 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7141 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     7201 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     7261 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gc
//
