LOCUS       sequence-491692-        2950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 driven gRNA expression of H1-g3 for targeting Dn29 attH1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2950)
  AUTHORS   Fanton A, Bartie LJ, Martins JQ, Tran VQ, Goudy L, Kernick C, 
            Durrant MG, Wei J, Armour-Garb Z, Pawluk A, Konermann S, Marson A, 
            Gilbert LA, Roth TL, Hsu PD
  TITLE     Site-specific DNA insertion into the human genome with engineered 
            recombinases.
  JOURNAL   Nat Biotechnol. 2025 Nov 6. doi: 10.1038/s41587-025-02895-3.
  PUBMED    41199024
REFERENCE   2  (bases 1 to 2950)
  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: "Nat 
            Biotechnol. 2025 Nov 6. doi: 10.1038/s41587-025-02895-3."
COMMENT     Sequence Label: pU6_H1-g3
FEATURES             Location/Qualifiers
     source          1..2950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..255
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7..27
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     178..197
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        276..351
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      433..888
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(520..539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     730..751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(905..924)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1024..1046
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1084..1102)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1170..1274
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1275..1343,1344..2135)
                     /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     complement(1493..1512)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2306..2894
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2795..2814
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctagcgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
       61 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      121 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      181 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      241 aaaggacgaa acaccagtgt gtattctgct gttgtgtttt agagctagaa atagcaagtt
      301 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttga
      361 attctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
      421 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
      481 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
      541 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      601 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
      661 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
      721 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
      781 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
      841 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
      901 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
      961 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     1021 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     1081 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     1141 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1201 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1261 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1321 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1381 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1441 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1501 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1561 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1621 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1681 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1741 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1801 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1861 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1921 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1981 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2041 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2101 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2161 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2221 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2281 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2341 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2401 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2461 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2521 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2581 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2641 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2701 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2761 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2821 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2881 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2941 cttttgctca
//
