LOCUS       sequence-182688-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human ESPL1.
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: ESPL1 D7.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    124..145
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(160..165,166..183,184..190)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    198..214
                     /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     203..225
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     222..238
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     222..238
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     257..277
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        259..277
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        305..531
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             532..712
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    759..884
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1377..1610
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1795..1839
                     /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        2017..2265
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2017..2037
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2188..2207
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2420..2537
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2606..2817
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2843..3442
                     /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(2843..2862)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3339..3359
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3458..4046
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3511..3531)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3929..3940)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4118..4351
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4429..4550
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4466..4485)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4520..4539
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4590..4725
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4745..4764)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4746..4764)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4774..4791)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4774..4790)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4783..4805)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4932..5387
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5019..5038)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5229..5250
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5413..5517
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5518..5586,5587..6378)
                     /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(5736..5755)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6549..7137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7038..7057
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7291..7301,1..7)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
       61 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
      121 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
      181 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
      241 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc
      301 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
      361 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      421 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      481 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      541 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      601 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      661 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      721 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      781 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      841 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      901 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      961 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
     1021 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
     1081 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
     1141 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
     1201 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
     1261 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1321 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1381 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1441 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1501 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1561 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1621 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1681 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1741 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1801 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1861 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1921 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1981 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     2041 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     2101 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     2161 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     2221 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgctag cttgccctag
     2281 gcatcgttta agagctatgc tggaaacagc atagcaagtt taaataaggc tagtccgtta
     2341 tcaacttgaa aaagtggcac cgagtcggtg ctttttttga attctagatc ttgagacaaa
     2401 tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg
     2461 aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta
     2521 caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc
     2581 tcgagtggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     2641 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
     2701 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     2761 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg
     2821 tcgtgacgcg ggatccgcca ccatgaccga gtacaagccc acggtgcgcc tcgccacccg
     2881 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     2941 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     3001 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     3061 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     3121 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     3181 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga
     3241 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     3301 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     3361 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     3421 gacccgcaag cccggtgcct gaacgcgtta agtcgacaat caacctctgg attacaaaat
     3481 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     3541 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     3601 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     3661 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     3721 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     3781 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     3841 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     3901 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     3961 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     4021 gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct
     4081 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     4141 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     4201 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     4261 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     4321 cccttttagt cagtgtggaa aatctctagc agtacgtata gtagttcatg tcatcttatt
     4381 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt
     4441 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4501 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     4561 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4621 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     4681 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa
     4741 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     4801 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4861 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4921 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4981 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     5041 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     5101 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     5161 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     5221 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     5281 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     5341 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     5401 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5461 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5521 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5581 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5641 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5701 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5761 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5821 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5881 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5941 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     6001 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6061 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6121 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6181 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6241 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6301 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6361 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6421 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6481 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6541 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6601 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6661 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6721 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6781 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6841 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6901 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6961 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7021 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7081 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7141 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     7201 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     7261 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag t
//
