LOCUS       sequence-387839-        7747 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Mettl3-targeting sgRNA driven by the U6 promoter and 
            Cre-recombinase driven by the PGK promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7747)
  AUTHORS   Raj N, Wang M, Seoane JA, Zhao RL, Kaiser AM, Moonie NA, Demeter J, 
            Boutelle AM, Kerr CH, Mulligan AS, Moffatt C, Zeng SX, Lu H, Barna 
            M, Curtis C, Chang HY, Jackson PK, Attardi LD
  TITLE     The Mettl3 epitranscriptomic writer amplifies p53 stress responses.
  JOURNAL   Mol Cell. 2022 Jul 7;82(13):2370-2384.e10. doi: 
            10.1016/j.molcel.2022.04.010. Epub 2022 May 4.
  PUBMED    35512709
REFERENCE   2  (bases 1 to 7747)
  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.molcel.2022.04.010"; journalName: "Mol Cell"; date: 
            "2022-07-7- 7"; volume: "82"; issue: "13"; pages: "2370-2384.e10"
COMMENT     Sequence Label: Lenti-U6-sgMettl3
FEATURES             Location/Qualifiers
     source          1..7747
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(760..1551,1552..1620))
                     /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     1383..1402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1621..1725)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1800..1819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1991..2370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2372..2570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2525..2545
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2588..2768
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2815..2940
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3437..3670
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3855..3899
                     /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    4197..4314
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4396..4644
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4396..4416
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4567..4586
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4665..4740
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4793..5280
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4828..4851)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5213..5232
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             5284..5304
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5305..6333
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     misc_feature    6389..6977
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6442..6462)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6860..6871)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6980..6996)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6981..6997)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             7506..7686
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      721 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      781 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
      841 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
      901 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
      961 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1021 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1081 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1141 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1201 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1261 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1321 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1381 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1441 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1501 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1561 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     1621 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1681 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1741 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     1801 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     1861 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     1921 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     1981 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     2041 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     2101 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     2161 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     2221 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     2281 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     2341 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     2401 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     2461 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     2521 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc
     2581 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     2641 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     2701 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     2761 ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct
     2821 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg
     2881 agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc
     2941 agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga
     3001 aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca
     3061 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa
     3121 ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc
     3181 tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag
     3241 gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg atcttcagac
     3301 ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa
     3361 aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa
     3421 aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta
     3481 tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc
     3541 agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag
     3601 tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc
     3661 aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt
     3721 ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg
     3781 agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc
     3841 aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt
     3901 ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag
     3961 gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc
     4021 agggatattc accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc
     4081 ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga
     4141 acggatcggc actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt
     4201 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     4261 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     4321 tattacaggg acagcagaga tccagtttgg ttagtaccgg gcccgctcta gccctgcagg
     4381 aactgtttaa actgtgaggg cctatttccc atgattcctt catatttgca tatacgatac
     4441 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
     4501 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     4561 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     4621 atcttgtgga aaggaccaaa caccggctta gggccgctag aggtgtttta gagctagaaa
     4681 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     4741 ttttttgttt ggcgcgccta tggcttaatt aacatggcgg ccgcattcta ccgggtaggg
     4801 gaggcgcttt tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg
     4861 cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg
     4921 gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc
     4981 ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact
     5041 agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc
     5101 ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc
     5161 gggggngggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc
     5221 acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggacccggg
     5281 atgcccaaga agaagaggaa ggtgtccaat ttactgaccg tacaccaaaa tttgcctgca
     5341 ttaccggtcg atgcaacgag tgatgaggtt cgcaagaacc tgatggacat gttcagggat
     5401 cgccaggcgt tttctgagca tacctggaaa atgcttctgt ccgtttgccg gtcgtgggcg
     5461 gcatggtgca agttgaataa ccggaaatgg tttcccgcag aacctgaaga tgttcgcgat
     5521 tatcttctat atcttcaggc gcgcggtctg gcagtaaaaa ctatccagca acatttgggc
     5581 cagctaaaca tgcttcatcg tcggtccggg ctgccacgac caagtgacag caatgctgtt
     5641 tcactggtta tgcggcggat ccgaaaagaa aacgttgatg ccggtgaacg tgcaaaacag
     5701 gctctagcgt tcgaacgcac tgatttcgac caggttcgtt cactcatgga aaatagcgat
     5761 cgctgccagg atatacgtaa tctggcattt ctggggattg cttataacac cctgttacgt
     5821 atagccgaaa ttgccaggat cagggttaaa gatatctcac gtactgacgg tgggagaatg
     5881 ttaatccata ttggcagaac gaaaacgctg gttagcaccg caggtgtaga gaaggcactt
     5941 agcctggggg taactaaact ggtcgagcga tggatttccg tctctggtgt agctgatgat
     6001 ccgaataact acctgttttg ccgggtcaga aaaaatggtg ttgccgcgcc atctgccacc
     6061 agccagctat caactcgcgc cctggaaggg atttttgaag caactcatcg attgatttac
     6121 ggcgctaagg atgactctgg tcagagatac ctggcctggt ctggacacag tgcccgtgtc
     6181 ggagccgcgc gagatatggc ccgcgctgga gtttcaatac cggagatcat gcaagctggt
     6241 ggctggacca atgtaaatat tgtcatgaac tatatccgta acctggatag tgaaacaggg
     6301 gcaatggtgc gcctgctgga agatggcgat tagccatgtt taagggttcc ggttccacta
     6361 ggtacaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag
     6421 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     6481 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     6541 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     6601 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     6661 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     6721 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     6781 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     6841 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     6901 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     6961 ttgggccgcc tccccgcatc gataccgtcg acctcgatcg agacctagaa aaacatggag
     7021 caatcacaag tagcaataca gcagctacca atgctgattg tgcctggcta gaagcacaag
     7081 aggaggagga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca atgacttaca
     7141 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     7201 actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc
     7261 ctgattggca gaactacaca ccagggccag ggatcagata tccactgacc tttggatggt
     7321 gctacaagct agtaccagtt gagcaagaga aggtagaaga agccaatgaa ggagagaaca
     7381 cccgcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtattag
     7441 agtggaggtt tgacagccgc ctagcatttc atcacatggc ccgagagctg catccggact
     7501 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     7561 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     7621 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     7681 ctagcagcat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     7741 tggcgtt
//
