LOCUS       sequence-450277-        9166 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRi guide RNA against MTCH2 (dual guide).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9166)
  AUTHORS   Guna A, Stevens TA, Inglis AJ, Replogle JM, Esantsi TK, Muthukumar 
            G, Shaffer KCL, Wang ML, Pogson AN, Jones JJ, Lomenick B, Chou TF, 
            Weissman JS, Voorhees RM
  TITLE     MTCH2 is a mitochondrial outer membrane protein insertase.
  JOURNAL   Science. 2022 Oct 21;378(6617):317-322. doi: 
            10.1126/science.add1856. Epub 2022 Oct 20.
  PUBMED    36264797
REFERENCE   2  (bases 1 to 9166)
  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: 
            "10.1126/science.add1856"; journalName: "Science"; date: 
            "2022-10-21- 21"; volume: "378"; issue: "6617"; pages: "317-322"
COMMENT     Sequence Label: pAG491
FEATURES             Location/Qualifiers
     source          1..9166
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    293..526
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             711..755
                     /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"
     misc_feature    1053..1170
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(1423..1456)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1460..1480
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     1705..1725
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1876..1895
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        2767..3945
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2998..3936
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3893..3913
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3957..4556
                     /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(3957..3976)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4453..4473
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(4575..4608)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4664..5252
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4717..4737)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5135..5146)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5255..5271)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5256..5272)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5781..5961
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6023..6611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6103..6122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6782..7573,7574..7642))
                     /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     7405..7424
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7643..7747)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7822..7841)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        8013..8392
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8394..8592
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8547..8567
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             8610..8790
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    8837..8962
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
ORIGIN
        1 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
       61 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
      121 accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca
      181 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
      241 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
      301 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
      361 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
      421 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
      481 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
      541 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
      601 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
      661 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
      721 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
      781 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
      841 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
      901 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
      961 gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg cgtgcgccaa
     1021 ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
     1081 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     1141 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     1201 gtttggttag taccgggccc gctctagaga tccgacgcgc catctctagg cccgcgccgg
     1261 ccccctcgca cggacttgtg ggagaagctc ggctactccc ctgccccggt taatttgcat
     1321 ataatatttc ctagtaacta tagaggctta atgtgcgata aaagacagat aatctgttct
     1381 ttttaatact agctacattt tacatgatag gcttggattt ctataacttc gtatagcata
     1441 cattatacga agttataaac agcacaaaag gaaactcacc ctaactgtaa agtaattgtg
     1501 tgttttgaga ctataagtat cccttggaga accaccttgt tggaccggct caccgggtcg
     1561 ctgtttcaga gctaagcaca agagtgcata gcaagttgaa ataaggctag tccgtttaca
     1621 acttgaaaaa gtggcacccg agtcgggtgc tttttttgct cgaatctaca ctcagctatg
     1681 gcgcgcccca aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat
     1741 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
     1801 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     1861 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     1921 gctttatata tcttgtggaa agccagaaac atggggctca ccgggtcgct tgggtttaag
     1981 agctaagctg gaaacagcat agcaagttta aataaggcta gtccgttatc aacttgaaaa
     2041 agtggcaccg agtcggtgct ttttttctcg agtactagga tccattaggc ggccgccggg
     2101 aggtggtccc tgcagttacg ccaatgataa cccccgccag aaaaatctta gtagccttcc
     2161 ctttttgttt tccgtgcccc aactcggcgg attgactcgg ccccttccgg aaacacccga
     2221 atcaacttct agtcaaatta ttgttcacgc cgcaatgacc cacccctggc ccgcgtctgt
     2281 ggaactgacc cctggtgtac aggagagttc gctgctgaaa gtggtcccaa aggggtacta
     2341 gtttttaagc tcccaactcc ccctccccca gcgtctggag gattccacac cctcgcaccg
     2401 caggggcgag gaagtgggcg gagtccggtt ttggcgccag ccgctgaggc tgccaagcag
     2461 aaaagccacc gctgaggaga ctccggtcac tgtcctcgcc ccgcctcccc cttccctccc
     2521 cttggggacc accgggcgcc acgccgcgaa cggtaagtgc cgcggtcgtc ggcgcctccg
     2581 ccctccccct agggccccaa ttcccagcgg gcgcggcgcg cggcccctcc ccccgccggg
     2641 cgcgcgcccg ctgccccgcc cttcgtggcc gcccggcgtg ggcggtgcca cccctccccc
     2701 cggcggcccc gcgcgcagct cccggctccc tcccccttcg gatgtggctt gagctgtagg
     2761 cgcggagatg catgtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     2821 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
     2881 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     2941 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta
     3001 agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct
     3061 tgaattactt ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt
     3121 gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag
     3181 gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc
     3241 gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt
     3301 ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt
     3361 ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc
     3421 ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg
     3481 gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg
     3541 gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa
     3601 tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc
     3661 tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac
     3721 ctcgattagt tctcgcgctt ttggagtacg tcgtctttag gttgggggga ggggttttat
     3781 gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg
     3841 atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct
     3901 cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgagctag cccaccatga
     3961 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca
     4021 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac ccggaccgcc
     4081 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     4141 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     4201 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     4261 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     4321 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     4381 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     4441 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     4501 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctaaaatt
     4561 cgtcgaggga cctaataact tcgtatagca tacattatac gaagttatac atgtttaagg
     4621 gttccggttc cactaggtac aattcgatat caagcttatc gataatcaac ctctggatta
     4681 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     4741 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     4801 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     4861 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     4921 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     4981 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     5041 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     5101 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     5161 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     5221 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgtcgacctc gatcgagacc
     5281 tagaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct
     5341 ggctagaagc acaagaggag gaggaggtgg gttttccagt cacacctcag gtacctttaa
     5401 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     5461 tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca
     5521 cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc agatatccac
     5581 tgacctttgg atggtgctac aagctagtac cagttgagca agagaaggta gaagaagcca
     5641 atgaaggaga gaacacccgc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
     5701 agagagaagt attagagtgg aggtttgaca gccgcctagc atttcatcac atggcccgag
     5761 agctgcatcc ggactgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     5821 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     5881 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     5941 cagtgtggaa aatctctagc agcatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     6001 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     6061 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     6121 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     6181 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     6241 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     6301 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6361 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     6421 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     6481 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6541 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6601 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6661 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6721 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6781 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6841 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6901 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6961 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     7021 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     7081 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     7141 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     7201 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     7261 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     7321 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     7381 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     7441 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7501 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7561 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7621 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7681 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7741 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     7801 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     7861 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     7921 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
     7981 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
     8041 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     8101 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     8161 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     8221 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     8281 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     8341 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     8401 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     8461 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     8521 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     8581 ctatataagc agcgcgtttt gcctgtactg ggtctctctg gttagaccag atctgagcct
     8641 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     8701 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     8761 ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa
     8821 agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag
     8881 aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga
     8941 gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa
     9001 aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag
     9061 cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag
     9121 acaaatactg ggacagctac aaccatccct tcagacagga tcagaa
//
