LOCUS       sequence-170485-        4416 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid vector encoding for U6-driven AAT_g2 gRNA (Z-allele 
            specific).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4416)
  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 4416)
  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_g2 gRNA
FEATURES             Location/Qualifiers
     source          1..4416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        23..271
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     23..43
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     194..213
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        292..367
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(379..403)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(497..513)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(497..513)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(510..532)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    521..537
                     /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(545..551,552..569,570..575))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    590..611
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(728..745)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(899..1487)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(979..998)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1658..2449,2450..2518))
                     /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     2281..2300
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(2536..3330)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2647..2666)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3257..3276
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(3674..4102)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3784..3805)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3996..4015
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4228..4250
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4242..4259
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4243..4259
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4266..4285
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4266..4284
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cggtaccaag gtcgggcagg aagagggcct atttcccatg attccttcat atttgcatat
       61 acgatacaag gctgttagag agataattag aattaatttg actgtaaaca caaagatatt
      121 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      181 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      241 tttatatatc ttgtggaaag gacgaaacac cgtgctgacc atcgacaaga agttttagag
      301 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      361 tcggtgcttt ttttctagac ccagctttct tgtacaaagt tggcattaac ctttgtcttc
      421 aataagggcg aattccagca cactggcggc cgttactagt ggatccgagc tcggtaccaa
      481 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
      541 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
      601 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
      661 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
      721 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
      781 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
      841 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
      901 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
      961 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     1021 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     1081 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     1141 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     1201 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     1261 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     1321 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     1381 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     1441 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     1501 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     1561 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     1621 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     1681 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     1741 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     1801 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     1861 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     1921 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     1981 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     2041 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     2101 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     2161 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     2221 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     2281 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     2341 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     2401 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     2461 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     2521 tcttcctttt tcaattcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga
     2581 atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc
     2641 ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg
     2701 gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc
     2761 atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa
     2821 cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc
     2881 ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca
     2941 ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc
     3001 ggcaggagca aggtgggatg acaggagatc ctgccccggc acttcgccca atagcagcca
     3061 gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc
     3121 cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt
     3181 cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca
     3241 gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga
     3301 acctgcgtgc aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc
     3361 agatcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt
     3421 gcagggcttc ccaaccttac cagagggcgc cccagctggc aattccggtt cgcttgctgt
     3481 ccataaaacc gcccagtcta gctatcgcca tgtaagccca ctgcaagcta cctgctttct
     3541 ctttgcgctt gcgttttccc ttgtccagat agcccagtag ctgacattca tccggggtca
     3601 gcaccgtttc tgcggactgg ctttctacgt gttccgcttc ctttagcagc ccttgcgccc
     3661 tgaattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     3721 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     3781 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     3841 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     3901 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     3961 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     4021 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     4081 aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc
     4141 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     4201 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     4261 aattgtaata cgactcacta tagggcgaat tgggccctct agatgcatgc tcgagcggcc
     4321 gccagtgtga tggatatctg cagaattcgc ccttattgaa gacttaagct gtacaaaaaa
     4381 gcaggcttta aaggaaccaa ttcagtcgac tggatc
//
