LOCUS       sequence-183802-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human MELK.
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: MELK E5.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    105..126
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(141..146,147..164,165..171)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    179..195
                     /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     184..206
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     203..219
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     203..219
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     238..258
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        240..258
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        286..512
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             513..693
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    740..865
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1358..1591
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1776..1820
                     /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        1998..2246
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1998..2018
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2169..2188
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2402..2519
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2588..2799
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2825..3424
                     /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(2825..2844)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3321..3341
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3440..4028
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3493..3513)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3911..3922)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4100..4333
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4411..4532
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4448..4467)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4502..4521
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4572..4707
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4727..4746)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4728..4746)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4756..4773)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4756..4772)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4765..4787)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4914..5369
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5001..5020)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5211..5232
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5395..5499
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5500..5568,5569..6360)
                     /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(5718..5737)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6531..7119
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7020..7039
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7273..7290
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
       61 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
      121 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
      181 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
      241 attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa
      301 tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga
      361 aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa
      421 ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata
      481 ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag
      541 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
      601 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
      661 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
      721 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
      781 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
      841 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
      901 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
      961 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
     1021 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
     1081 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
     1141 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     1201 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
     1261 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
     1321 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
     1381 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
     1441 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
     1501 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
     1561 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
     1621 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
     1681 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
     1741 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
     1801 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
     1861 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
     1921 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
     1981 ccccgagggg acccagagag ggcctatttc ccatgattcc ttcatatttg catatacgat
     2041 acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag atattagtac
     2101 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     2161 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     2221 atatcttgtg gaaaggacga aacaccgctc tccagtaagg atatggcgtt taagagctat
     2281 gctggaaaca gcatagcaag tttaaataag gctagtccgt tatcaacttg aaaaagtggc
     2341 accgagtcgg tgcttttttt gaattctaga tcttgagaca aatggcagta ttcatccaca
     2401 attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
     2461 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc
     2521 gggtttatta cagggacagc agagatccac tttggcgccg gctcgagtgg ctccggtgcc
     2581 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
     2641 aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
     2701 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
     2761 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg tgtcgtgacg cgggatccgc
     2821 caccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     2881 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     2941 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     3001 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     3061 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     3121 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     3181 ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct
     3241 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     3301 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     3361 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     3421 ctgaacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     3481 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3541 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3601 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     3661 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     3721 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     3781 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     3841 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     3901 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     3961 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     4021 ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt
     4081 ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt
     4141 tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     4201 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     4261 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     4321 aaaatctcta gcagtacgta tagtagttca tgtcatctta ttattcagta tttataactt
     4381 gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac
     4441 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4501 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc
     4561 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     4621 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     4681 agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc
     4741 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     4801 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     4861 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     4921 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4981 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     5041 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     5101 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     5161 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     5221 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     5281 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     5341 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     5401 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5461 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5521 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5581 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5641 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5701 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5761 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5821 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5881 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5941 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6001 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6061 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6121 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6181 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     6241 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6301 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6361 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6421 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6481 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6541 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6601 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6661 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6721 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6781 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6841 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6901 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6961 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7021 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7081 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7141 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     7201 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     7261 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag ag
//
