LOCUS       sequence-183631-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human NAE1.
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: NAE1 G8.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    133..154
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(169..174,175..192,193..199)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    207..223
                     /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     212..234
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     231..247
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     231..247
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     266..286
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        268..286
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        314..540
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             541..721
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    768..893
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1386..1619
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1804..1848
                     /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        2026..2274
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2026..2046
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2197..2216
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2429..2546
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2615..2826
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2852..3451
                     /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(2852..2871)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3348..3368
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3467..4055
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3520..3540)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3938..3949)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4127..4360
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4438..4559
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4475..4494)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4529..4548
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4599..4734
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4754..4773)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4755..4773)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4783..4800)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4783..4799)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4792..4814)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4941..5396
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5028..5047)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5238..5259
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5422..5526
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5527..5595,5596..6387)
                     /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(5745..5764)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6558..7146
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7047..7066
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7300..7301,1..16)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
       61 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
      121 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
      181 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
      241 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga
      301 gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg
      361 agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta
      421 aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga
      481 accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac
      541 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
      601 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      661 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      721 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      781 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      841 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      901 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      961 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
     1021 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
     1081 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
     1141 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
     1201 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
     1261 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     1321 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     1381 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     1441 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     1501 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1561 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1621 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1681 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1741 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1801 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1861 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1921 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1981 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagagaggg cctatttccc
     2041 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
     2101 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     2161 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     2221 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggttcg
     2281 tttacaatta ttgagtttaa gagctatgct ggaaacagca tagcaagttt aaataaggct
     2341 agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttgaa ttctagatct
     2401 tgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca
     2461 gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa
     2521 aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt
     2581 ggcgccggct cgagtggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc
     2641 cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg
     2701 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     2761 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     2821 acacaggtgt cgtgacgcgg gatccgccac catgaccgag tacaagccca cggtgcgcct
     2881 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     2941 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     3001 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     3061 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     3121 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     3181 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt
     3241 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     3301 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     3361 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     3421 ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga
     3481 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     3541 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     3601 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     3661 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     3721 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     3781 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     3841 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     3901 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     3961 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     4021 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac
     4081 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     4141 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
     4201 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     4261 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     4321 tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag tagttcatgt
     4381 catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac
     4441 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4501 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4561 catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca
     4621 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     4681 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga
     4741 cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca
     4801 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     4861 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     4921 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4981 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     5041 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     5101 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     5161 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     5221 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     5281 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     5341 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     5401 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5461 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5521 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5581 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5641 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5701 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5761 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5821 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5881 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5941 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     6001 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     6061 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     6121 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6181 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6241 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6301 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6361 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6421 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6481 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6541 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6601 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6661 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6721 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6781 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6841 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6901 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6961 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     7021 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     7081 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7141 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7201 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     7261 tgagctgata ccgctcgccg cagccgaacg accgagcgca g
//
