LOCUS       sequence-183922-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human RCC1.
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: RCC1 G3.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    87..108
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(123..128,129..146,147..153)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    161..177
                     /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     166..188
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     185..201
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     185..201
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     220..240
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        222..240
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        268..494
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             495..675
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    722..847
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1340..1573
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1758..1802
                     /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        1980..2228
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1980..2000
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2151..2170
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2384..2501
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2570..2781
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2807..3406
                     /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(2807..2826)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3303..3323
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3422..4010
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3475..3495)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3893..3904)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4082..4315
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4393..4514
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4430..4449)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4484..4503
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4554..4689
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4709..4728)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4710..4728)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4738..4755)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4738..4754)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4747..4769)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4896..5351
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4983..5002)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5193..5214
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5377..5481
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5482..5550,5551..6342)
                     /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(5700..5719)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6513..7101
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7002..7021
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7255..7272
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
       61 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
      121 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
      181 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
      241 gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca
      301 acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc
      361 gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga
      421 catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag
      481 ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct
      541 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
      601 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
      661 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
      721 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
      781 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
      841 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
      901 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
      961 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
     1021 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
     1081 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
     1141 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca
     1201 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
     1261 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
     1321 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
     1381 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
     1441 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
     1501 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
     1561 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
     1621 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
     1681 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
     1741 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
     1801 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag
     1861 taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta
     1921 ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccagag
     1981 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     2041 taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     2101 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     2161 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     2221 gaaacaccga cactaggcca gggcgacgtg tttaagagct atgctggaaa cagcatagca
     2281 agtttaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     2341 ttgaattcta gatcttgaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2401 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2461 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2521 gcagagatcc actttggcgc cggctcgagt ggctccggtg cccgtcagtg ggcagagcgc
     2581 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag
     2641 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc
     2701 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac
     2761 gggtttgccg ccagaacaca ggtgtcgtga cgcgggatcc gccaccatga ccgagtacaa
     2821 gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc
     2881 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg
     2941 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     3001 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     3061 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     3121 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     3181 ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     3241 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc
     3301 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     3361 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga
     3421 caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     3481 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     3541 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     3601 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     3661 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     3721 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     3781 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     3841 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     3901 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     3961 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta
     4021 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     4081 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct
     4141 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     4201 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     4261 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg
     4321 tatagtagtt catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca
     4381 gagagtgaga ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4441 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4501 atcaatgtat cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc
     4561 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4621 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     4681 ttggaggcct agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     4741 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     4801 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     4861 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag
     4921 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     4981 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5041 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5101 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5161 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5221 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     5281 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     5341 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5401 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5461 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5521 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5581 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5641 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5701 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5761 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5821 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5881 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5941 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6001 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     6061 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6121 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6181 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6241 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6301 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6361 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6421 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6481 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6541 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6601 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6661 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6721 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6781 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6841 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6901 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6961 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     7021 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7081 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     7141 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     7201 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     7261 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cg
//
