LOCUS       sequence-170254-        2905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid vector encoding for U6-driven AAT_g1 gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2905)
  AUTHORS   Eggenschwiler R, Moslem M, Fraguas MS, Galla M, Papp O, Naujock M, 
            Fonfara I, Gensch I, Wahner A, Beh-Pajooh A, Mussolino C, Tauscher 
            M, Steinemann D, Wegner F, Petri S, Schambach A, Charpentier E, 
            Cathomen T, Cantz T
  TITLE     Improved bi-allelic modification of a transcriptionally silent locus
            in patient-derived iPSC by Cas9 nickase.
  JOURNAL   Sci Rep. 2016 Dec 2;6:38198. doi: 10.1038/srep38198.
  PUBMED    27910942
REFERENCE   2  (bases 1 to 2905)
  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"; journalName: "Sci Rep."; 
            date: "2016-12-2"; volume: "6:38198. doi"; pages: " 
            10.1038/srep38198"
COMMENT     Sequence Label: AAT_g1 gRNA
FEATURES             Location/Qualifiers
     source          1..2905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        70..318
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     70..90
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     241..260
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        339..414
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(426..450)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(485..507)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    496..512
                     /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(520..526,527..544,545..550))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    565..586
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(703..720)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(874..1462)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(954..973)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1633..2424,2425..2493))
                     /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     2256..2275
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2494..2598)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2666..2684
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2722..2744)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2844..2863
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgtacaaaaa agcaggcttt aaaggaacca attcagtcga ctggatccgg taccaaggtc
       61 gggcaggaag agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
      121 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
      181 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
      241 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
      301 tggaaaggac gaaacaccgc acagccttat gcacggccgt tttagagcta gaaatagcaa
      361 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
      421 tctagaccca gctttcttgt acaaagttgg cattaacctg cggccgcaag cttggatccg
      481 aattcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
      541 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
      601 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
      661 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
      721 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
      781 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
      841 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
      901 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
      961 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1021 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     1081 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1141 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1201 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1261 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     1321 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     1381 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     1441 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     1501 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     1561 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     1621 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     1681 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     1741 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     1801 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     1861 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     1921 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     1981 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     2041 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     2101 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     2161 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     2221 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     2281 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     2341 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     2401 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     2461 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     2521 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2581 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     2641 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     2701 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     2761 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     2821 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     2881 gggtaccgag ctcgcggccg caagc
//
