LOCUS       sequence-183800-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human NDEL1.
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: NDEL1 F3.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    44..65
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(80..85,86..103,104..110)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    118..134
                     /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     123..145
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     142..158
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     142..158
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     177..197
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        179..197
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        225..451
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             452..632
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    679..804
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1297..1530
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1715..1759
                     /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        1937..2185
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1937..1957
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2108..2127
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2340..2457
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2526..2737
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2763..3362
                     /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(2763..2782)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3259..3279
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3378..3966
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3431..3451)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3849..3860)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4038..4271
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4349..4470
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4386..4405)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4440..4459
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4510..4645
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4665..4684)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4666..4684)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4694..4711)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4694..4710)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4703..4725)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4852..5307
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4939..4958)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5149..5170
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5333..5437
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5438..5506,5507..6298)
                     /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(5656..5675)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6469..7057
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6958..6977
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7211..7228
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
       61 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
      121 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa
      181 ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt cttatgcaat
      241 actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac aaggagagaa
      301 aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct tattaggaag
      361 gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt gcagagatat
      421 tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc agatctgagc
      481 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
      541 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
      601 acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg
      661 aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca
      721 agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag
      781 gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa
      841 aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca
      901 agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt
      961 agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca
     1021 ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc
     1081 aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa
     1141 gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt
     1201 atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag
     1261 aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt
     1321 gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca
     1381 attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca
     1441 gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt
     1501 ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat
     1561 ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg
     1621 gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca
     1681 ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt
     1741 agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa
     1801 attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc
     1861 tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc acctcccaac
     1921 cccgagggga cccagagagg gcctatttcc catgattcct tcatatttgc atatacgata
     1981 caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca
     2041 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     2101 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     2161 tatcttgtgg aaaggacgaa acaccgttgg aggcacaatt agtacgttta agagctatgc
     2221 tggaaacagc atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac
     2281 cgagtcggtg ctttttttga attctagatc ttgagacaaa tggcagtatt catccacaat
     2341 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata
     2401 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg
     2461 gtttattaca gggacagcag agatccactt tggcgccggc tcgagtggct ccggtgcccg
     2521 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
     2581 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
     2641 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
     2701 cgttcttttt cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg ggatccgcca
     2761 ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg
     2821 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg
     2881 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     2941 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     3001 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     3061 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     3121 agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg
     3181 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     3241 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     3301 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct
     3361 gaacgcgtta agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     3421 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     3481 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     3541 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     3601 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     3661 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3721 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     3781 cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     3841 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     3901 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     3961 ccccgcgtcg actttaagac caatgactta caaggcagct gtagatctta gccacttttt
     4021 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt
     4081 tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     4141 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     4201 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     4261 aatctctagc agtacgtata gtagttcatg tcatcttatt attcagtatt tataacttgc
     4321 aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa
     4381 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4441 tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta
     4501 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4561 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     4621 tagtgaggag gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt
     4681 attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     4741 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     4801 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct
     4861 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     4921 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     4981 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5041 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     5101 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5161 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     5221 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5281 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     5341 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5401 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     5461 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     5521 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     5581 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     5641 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     5701 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     5761 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     5821 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     5881 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     5941 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6001 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6061 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6121 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     6181 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     6241 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     6301 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     6361 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6421 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     6481 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     6541 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     6601 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     6661 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     6721 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     6781 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     6841 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     6901 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     6961 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7021 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7081 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     7141 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     7201 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     7261 tctccccgcg cgttggccga ttcattaatg cagctggcac g
//
