LOCUS       sequence-183409-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human SMC3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7301)
  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 7301)
  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: SMC3 A7.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    128..149
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(164..169,170..187,188..194)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    202..218
                     /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     207..229
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     226..242
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     226..242
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     261..281
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        263..281
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        309..535
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             536..716
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    763..888
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1381..1614
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1799..1843
                     /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        2021..2269
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2021..2041
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2192..2211
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2424..2541
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2610..2821
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2847..3446
                     /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(2847..2866)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3343..3363
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3462..4050
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3515..3535)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3933..3944)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4122..4355
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4433..4554
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4470..4489)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4524..4543
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4594..4729
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4749..4768)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4750..4768)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4778..4795)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4778..4794)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4787..4809)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4936..5391
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5023..5042)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5233..5254
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5417..5521
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5522..5590,5591..6382)
                     /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(5740..5759)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6553..7141
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7042..7061
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7295..7301,1..11)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
       61 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      121 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      181 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
      241 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc
      301 aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
      361 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
      421 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
      481 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc
      541 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      601 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      661 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      721 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
      781 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
      841 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
      901 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
      961 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     1021 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     1081 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     1141 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     1201 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     1261 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     1321 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     1381 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     1441 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     1501 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     1561 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     1621 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     1681 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     1741 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     1801 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     1861 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     1921 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     1981 atcgtttcag acccacctcc caaccccgag gggacccaga gagggcctat ttcccatgat
     2041 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac
     2101 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     2161 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     2221 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gataaaatga
     2281 gacgagcccg tttaagagct atgctggaaa cagcatagca agtttaaata aggctagtcc
     2341 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgaattcta gatcttgaga
     2401 caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca
     2461 ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa
     2521 attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggcgc
     2581 cggctcgagt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
     2641 gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa
     2701 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
     2761 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
     2821 ggtgtcgtga cgcgggatcc gccaccatga ccgagtacaa gcccacggtg cgcctcgcca
     2881 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     2941 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     3001 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     3061 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     3121 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     3181 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc
     3241 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     3301 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     3361 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     3421 gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct ctggattaca
     3481 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3541 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3601 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3661 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3721 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3781 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3841 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     3901 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     3961 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     4021 gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc
     4081 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     4141 ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg
     4201 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     4261 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     4321 cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt catgtcatct
     4381 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt
     4441 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4501 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4561 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4621 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4681 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac
     4741 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     4801 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     4861 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     4921 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4981 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     5041 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5101 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5161 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5221 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5281 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5341 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     5401 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5461 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5521 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5581 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5641 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5701 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5761 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5821 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5881 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5941 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6001 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6061 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6121 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6181 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6241 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6301 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6361 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6421 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6481 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6541 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6601 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6661 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6721 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6781 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6841 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6901 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6961 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7021 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7081 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7141 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7201 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     7261 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag t
//
