LOCUS       sequence-182960-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human RAD21.
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: RAD21 G12.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..36
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    153..174
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(189..194,195..212,213..219)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    227..243
                     /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     232..254
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     251..267
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     251..267
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     286..306
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        288..306
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        334..560
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             561..741
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    788..913
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1406..1639
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1824..1868
                     /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        2046..2294
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2046..2066
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2217..2236
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2450..2567
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2636..2847
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2873..3472
                     /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(2873..2892)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3369..3389
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3488..4076
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3541..3561)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3959..3970)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4148..4381
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4459..4580
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4496..4515)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4550..4569
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4620..4755
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4775..4794)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4776..4794)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4804..4821)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4804..4820)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4813..4835)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4962..5417
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5049..5068)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5259..5280
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5443..5547
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5548..5616,5617..6408)
                     /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(5766..5785)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6579..7167
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7068..7087
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
       61 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
      121 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
      181 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
      241 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
      301 taaagggaac aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt
      361 cttgcaacat ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg
      421 catgccgatt ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg
      481 gtctgacatg gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt
      541 gcctagctcg atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct
      601 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
      661 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
      721 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
      781 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
      841 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
      901 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
      961 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
     1021 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
     1081 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     1141 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     1201 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga
     1261 tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata
     1321 aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc
     1381 agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag
     1441 gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg
     1501 gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc
     1561 aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc
     1621 taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg
     1681 ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga
     1741 cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg
     1801 aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg
     1861 caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa
     1921 tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata
     1981 gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac
     2041 ccagagaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     2101 gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     2161 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     2221 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     2281 aaggacgaaa caccgtaaat tacactcgaa cacatgttta agagctatgc tggaaacagc
     2341 atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     2401 ctttttttga attctagatc ttgagacaaa tggcagtatt catccacaat tttaaaagaa
     2461 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
     2521 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
     2581 gggacagcag agatccactt tggcgccggc tcgagtggct ccggtgcccg tcagtgggca
     2641 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt
     2701 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
     2761 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
     2821 cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg ggatccgcca ccatgaccga
     2881 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     2941 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     3001 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     3061 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     3121 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     3181 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     3241 gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     3301 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     3361 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     3421 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta
     3481 agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     3541 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     3601 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     3661 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     3721 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     3781 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3841 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     3901 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3961 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     4021 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg
     4081 actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     4141 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact
     4201 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     4261 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     4321 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     4381 agtacgtata gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga
     4441 atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4501 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4561 aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca
     4621 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4681 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     4741 gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg
     4801 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     4861 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     4921 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     4981 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     5041 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5101 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5161 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5221 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5281 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     5341 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5401 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5461 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5521 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5581 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5641 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5701 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5761 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5821 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5881 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5941 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6001 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6061 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6121 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6181 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6241 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6301 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6361 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6421 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6481 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6541 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6601 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6661 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6721 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6781 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6841 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6901 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6961 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     7021 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     7081 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7141 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7201 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     7261 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gc
//
