LOCUS       sequence-190610-        7324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human SMC6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7324)
  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 7324)
  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: SMC6 G4.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    49..70
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(85..90,91..108,109..115)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    123..139
                     /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     128..150
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     147..163
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     147..163
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     182..202
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        184..202
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        230..456
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             457..637
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    684..809
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1302..1535
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1720..1764
                     /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        1942..2190
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1942..1962
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2113..2132
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2368..2485
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2554..2765
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2791..3390
                     /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(2791..2810)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3287..3307
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3406..3994
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3459..3479)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3877..3888)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4066..4299
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4377..4498
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4414..4433)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4468..4487
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4538..4673
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4693..4712)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4694..4712)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4722..4739)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4722..4738)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4731..4753)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4880..5335
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4967..4986)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5177..5198
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5361..5465
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5466..5534,5535..6326)
                     /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(5684..5703)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6497..7085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6986..7005
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7239..7256
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
       61 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      121 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
      181 cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat
      241 gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga
      301 gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta
      361 ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga
      421 gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc
      481 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      541 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      601 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      661 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      721 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      781 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      841 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      901 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      961 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
     1021 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
     1081 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
     1141 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     1201 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1261 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1321 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1381 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1441 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1501 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1561 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1621 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1681 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1741 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1801 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1861 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1921 ccaaccccga ggggacccag agagggccta tttcccatga ttccttcata tttgcatata
     1981 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
     2041 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     2101 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     2161 ttatatatct tgtggaaagg acgaaacacc gacagctctc atagtcggtc tgtttaagag
     2221 ctatgstsgr wmtgtttaag agctatgctg gaaacagcat agcaagttta aataaggcta
     2281 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttgaat tctagatctt
     2341 gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     2401 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     2461 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg
     2521 gcgccggctc gagtggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
     2581 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
     2641 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
     2701 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
     2761 cacaggtgtc gtgacgcggg atccgccacc atgaccgagt acaagcccac ggtgcgcctc
     2821 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     2881 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     2941 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     3001 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     3061 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     3121 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc
     3181 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     3241 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     3301 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     3361 tggtgcatga cccgcaagcc cggtgcctga acgcgttaag tcgacaatca acctctggat
     3421 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     3481 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     3541 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     3601 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     3661 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     3721 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3781 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     3841 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3901 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     3961 acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca
     4021 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     4081 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
     4141 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     4201 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     4261 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tacgtatagt agttcatgtc
     4321 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact
     4381 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4441 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4501 atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4561 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4621 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac
     4681 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     4741 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     4801 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     4861 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4921 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     4981 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     5041 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     5101 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     5161 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     5221 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     5281 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     5341 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5401 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5461 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5521 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5581 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5641 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5701 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5761 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5821 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5881 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5941 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6001 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6061 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6121 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6181 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6241 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6301 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6361 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6421 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6481 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6541 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6601 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6661 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6721 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6781 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6841 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6901 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     6961 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     7021 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7081 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7141 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     7201 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     7261 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     7321 gctg
//
