LOCUS       sequence-189914-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human NDUFAB1.
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: NDUFAB1 C3.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    94..115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(130..135,136..153,154..160)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    168..184
                     /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     173..195
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     192..208
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     192..208
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     227..247
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        229..247
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        275..501
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             502..682
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    729..854
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1347..1580
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1765..1809
                     /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        1987..2235
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1987..2007
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2158..2177
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2390..2507
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2576..2787
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2813..3412
                     /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(2813..2832)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3309..3329
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3428..4016
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3481..3501)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3899..3910)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4088..4321
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4399..4520
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4436..4455)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4490..4509
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4560..4695
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4715..4734)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4716..4734)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4744..4761)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4744..4760)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4753..4775)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4902..5357
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4989..5008)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5199..5220
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5383..5487
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5488..5556,5557..6348)
                     /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(5706..5725)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6519..7107
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7008..7027
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7261..7278
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
       61 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
      121 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
      181 acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca
      241 ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt cttatgcaat actcttgtag
      301 tcttgcaaca tggtaacgat gagttagcaa catgccttac aaggagagaa aaagcaccgt
      361 gcatgccgat tggtggaagt aaggtggtac gatcgtgcct tattaggaag gcaacagacg
      421 ggtctgacat ggattggacg aaccactgaa ttgccgcatt gcagagatat tgtatttaag
      481 tgcctagctc gatacataaa cgggtctctc tggttagacc agatctgagc ctgggagctc
      541 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
      601 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
      661 tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac
      721 cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg
      781 ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg
      841 ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt
      901 taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc
      961 tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac
     1021 tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata
     1081 cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt
     1141 tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg
     1201 atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat
     1261 aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg
     1321 cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca
     1381 ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct
     1441 ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg
     1501 caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac
     1561 ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact
     1621 gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg
     1681 acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt
     1741 gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg
     1801 gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa attattcata
     1861 atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat
     1921 agagttaggc agggatattc accattatcg tttcagaccc acctcccaac cccgagggga
     1981 cccagagagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
     2041 agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
     2101 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
     2161 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
     2221 aaaggacgaa acaccgcaaa gtcccgagcc tcgtcgttta agagctatgc tggaaacagc
     2281 atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     2341 ctttttttga attctagatc ttgagacaaa tggcagtatt catccacaat tttaaaagaa
     2401 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
     2461 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
     2521 gggacagcag agatccactt tggcgccggc tcgagtggct ccggtgcccg tcagtgggca
     2581 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt
     2641 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
     2701 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
     2761 cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg ggatccgcca ccatgaccga
     2821 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     2881 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     2941 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     3001 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     3061 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     3121 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     3181 gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     3241 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     3301 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     3361 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta
     3421 agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     3481 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     3541 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     3601 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     3661 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     3721 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3781 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     3841 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3901 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     3961 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg
     4021 actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     4081 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact
     4141 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     4201 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     4261 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     4321 agtacgtata gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga
     4381 atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4441 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4501 aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca
     4561 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4621 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     4681 gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg
     4741 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     4801 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     4861 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     4921 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4981 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5041 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5101 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5161 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5221 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     5281 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5341 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5401 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5461 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5521 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5581 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5641 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5701 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5761 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5821 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5881 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5941 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6001 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6061 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6121 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6181 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6241 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6301 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6361 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6421 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6481 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6541 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6601 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6661 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6721 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6781 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6841 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6901 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6961 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     7021 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7081 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7141 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     7201 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     7261 agcgagtcag tgagcgagga agcggaagag cgcccaatac g
//
