LOCUS       sequence-183201-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human KIF20A.
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: KIF20A D5.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    64..85
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(100..105,106..123,124..130)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    138..154
                     /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     143..165
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     162..178
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     162..178
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     197..217
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        199..217
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        245..471
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             472..652
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    699..824
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1317..1550
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1735..1779
                     /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        1957..2205
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1957..1977
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2128..2147
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2361..2478
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2547..2758
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2784..3383
                     /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(2784..2803)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3280..3300
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3399..3987
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3452..3472)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3870..3881)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4059..4292
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4370..4491
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4407..4426)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4461..4480
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4531..4666
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4686..4705)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4687..4705)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4715..4732)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4715..4731)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4724..4746)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4873..5328
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4960..4979)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5170..5191
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5354..5458
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5459..5527,5528..6319)
                     /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(5677..5696)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6490..7078
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6979..6998
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7232..7249
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
       61 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
      121 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
      181 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc
      241 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
      301 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      361 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      421 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      481 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      541 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      601 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      661 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      721 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      781 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      841 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      901 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      961 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
     1021 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
     1081 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
     1141 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
     1201 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1261 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1321 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1381 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1441 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1501 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1561 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1621 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1681 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1741 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1801 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1861 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1921 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1981 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     2041 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     2101 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     2161 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgagaa actacgacat
     2221 cgtcatgttt aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt
     2281 atcaacttga aaaagtggca ccgagtcggt gctttttttg aattctagat cttgagacaa
     2341 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     2401 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     2461 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg
     2521 ctcgagtggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
     2581 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg
     2641 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
     2701 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt
     2761 gtcgtgacgc gggatccgcc accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     2821 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     2881 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     2941 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     3001 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     3061 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     3121 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg
     3181 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     3241 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     3301 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     3361 tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa
     3421 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     3481 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     3541 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     3601 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     3661 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     3721 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     3781 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     3841 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     3901 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     3961 ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc
     4021 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     4081 acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc
     4141 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
     4201 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
     4261 acccttttag tcagtgtgga aaatctctag cagtacgtat agtagttcat gtcatcttat
     4321 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat
     4381 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4441 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4501 gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4561 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4621 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca
     4681 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg
     4741 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     4801 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     4861 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4921 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4981 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     5041 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     5101 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5161 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     5221 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     5281 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     5341 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5401 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5461 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5521 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5581 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5641 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5701 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5761 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5821 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5881 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5941 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     6001 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6061 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6121 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6181 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6241 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6301 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6361 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6421 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6481 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6541 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6601 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6661 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6721 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6781 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6841 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6901 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6961 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7021 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7081 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     7141 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     7201 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     7261 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg at
//
