LOCUS       sequence-183195-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human OIP5.
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: OIP5 B3.6 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     37..54
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    171..192
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(207..212,213..230,231..237)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    245..261
                     /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     250..272
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     269..285
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     269..285
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     304..324
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        306..324
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        352..578
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             579..759
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    806..931
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1424..1657
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1842..1886
                     /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        2064..2312
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2064..2084
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2235..2254
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2468..2585
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2654..2865
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2891..3490
                     /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(2891..2910)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3387..3407
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3506..4094
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3559..3579)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3977..3988)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4166..4399
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4477..4598
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4514..4533)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4568..4587
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4638..4773
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4793..4812)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4794..4812)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4822..4839)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4822..4838)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4831..4853)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4980..5435
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5067..5086)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5277..5298
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5461..5565
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5566..5634,5635..6426)
                     /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(5784..5803)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6597..7185
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7086..7105
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
       61 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
      121 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
      181 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
      241 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
      301 cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt
      361 atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag
      421 gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat
      481 taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca
      541 gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga
      601 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
      661 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
      721 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
      781 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
      841 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
      901 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
      961 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
     1021 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
     1081 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     1141 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     1201 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     1261 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     1321 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     1381 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     1441 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     1501 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     1561 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     1621 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     1681 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     1741 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     1801 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     1861 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     1921 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     1981 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc
     2041 tcccaacccc gaggggaccc agagagggcc tatttcccat gattccttca tatttgcata
     2101 tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat
     2161 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     2221 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     2281 ctttatatat cttgtggaaa ggacgaaaca ccgtcacgca gtgctcgccg actgtttaag
     2341 agctatgctg gaaacagcat agcaagttta aataaggcta gtccgttatc aacttgaaaa
     2401 agtggcaccg agtcggtgct ttttttgaat tctagatctt gagacaaatg gcagtattca
     2461 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     2521 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     2581 attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc gagtggctcc
     2641 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
     2701 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
     2761 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     2821 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc gtgacgcggg
     2881 atccgccacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     2941 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     3001 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     3061 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     3121 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     3181 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     3241 gcccaaggag cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa
     3301 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     3361 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     3421 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     3481 cggtgcctga acgcgttaag tcgacaatca acctctggat tacaaaattt gtgaaagatt
     3541 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     3601 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     3661 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     3721 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     3781 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     3841 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     3901 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     3961 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     4021 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     4081 ggccgcctcc ccgcgtcgac tttaagacca atgacttaca aggcagctgt agatcttagc
     4141 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     4201 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     4261 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     4321 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     4381 gtgtggaaaa tctctagcag tacgtatagt agttcatgtc atcttattat tcagtattta
     4441 taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat
     4501 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4561 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc
     4621 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     4681 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     4741 tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag
     4801 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     4861 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     4921 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     4981 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     5041 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5101 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5161 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     5221 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     5281 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     5341 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     5401 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     5461 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5521 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5581 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5641 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5701 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5761 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5821 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5881 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5941 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6001 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6061 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6121 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6181 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6241 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6301 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6361 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6421 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6481 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6541 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6601 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6661 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6721 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6781 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6841 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6901 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6961 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7021 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7081 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     7141 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     7201 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     7261 tcccctgatt ctgtggataa ccgtattacc gcctttgagt ga
//
