LOCUS       sequence-375286-        3027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a gRNA for base editing the EGFP Kozak sequence of 
            pWPT-/mEGFP-1T-IRES-mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3027)
  AUTHORS   Ambrosini C, Destefanis E, Kheir E, Broso F, Alessandrini F, Longhi 
            S, Battisti N, Pesce I, Dassi E, Petris G, Cereseto A, Quattrone A
  TITLE     Translational enhancement by base editing of the Kozak sequence 
            rescues haploinsufficiency.
  JOURNAL   Nucleic Acids Res. 2022 Oct 14;50(18):10756-10771. doi: 
            10.1093/nar/gkac799.
  PUBMED    36165847
REFERENCE   2  (bases 1 to 3027)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2022-10-14- 14"; volume: "50"; issue: 
            "18"; pages: "10756-10771"
COMMENT     Sequence Label: pUC19-sg-1
FEATURES             Location/Qualifiers
     source          1..3027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(32..54)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     154..173
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     367..389
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     381..398
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     382..398
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        405..653
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     405..425
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     576..595
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(809..825)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(809..825)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(822..844)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    833..849
                     /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)."
     promoter        complement(join(857..863,864..881,882..887))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    902..923
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1040..1057)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1211..1799)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1291..1310)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1970..2761,2762..2830))
                     /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     2593..2612
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2831..2935)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3003..3021
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
       61 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
      121 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
      181 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
      241 gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
      301 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      361 ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcgagggc ctatttccca
      421 tgattccttc atatttgcat atacgataca aggctgttag agagataatt ggaattaatt
      481 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
      541 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
      601 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgccatag
      661 tggcttagtt agatgtttaa gagctatgct ggaaacagca tagcaagttt aaataaggct
      721 agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttcta gagtcgacct
      781 gcaggcatgc aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
      841 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
      901 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
      961 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     1021 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     1081 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     1141 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     1201 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     1261 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     1321 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     1381 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     1441 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     1501 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     1561 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     1621 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     1681 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     1741 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     1801 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     1861 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     1921 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     1981 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     2041 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     2101 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     2161 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     2221 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     2281 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     2341 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     2401 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     2461 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     2521 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     2581 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     2641 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     2701 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     2761 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     2821 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     2881 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     2941 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     3001 aaaataggcg tatcacgagg ccctttc
//
