LOCUS       sequence-225628-        3180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of gRNA targeting TMTC4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3180)
  AUTHORS   Narimatsu Y, Joshi HJ, Zhang Y, Gomes C, Chen YH, Lorenzetti F, 
            Furukawa S, Schjoldager K, Hansen L, Clausen H, Bennett EP, Wandall 
            HH
  TITLE     A validated gRNA library for CRISPR/Cas9 targeting of the human 
            glycosyltransferase genome.
  JOURNAL   Glycobiology. 2018 Jan 5. pii: 4791732. doi: 10.1093/glycob/cwx101.
  PUBMED    29315387
REFERENCE   2  (bases 1 to 3180)
  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: 
            "Glycobiology. 2018 Jan 5. pii: 4791732. doi: 
            10.1093/glycob/cwx101."
COMMENT     Sequence Label: gh163
FEATURES             Location/Qualifiers
     source          1..3180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   443..583
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   443..572
                     /label=AAV2 ITR
     rep_origin      658..1113
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(745..764)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     955..976
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1130..1149)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1249..1271
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1309..1327)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1395..1499
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1500..1568,1569..2360)
                     /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(1718..1737)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2531..3119
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3020..3039
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ctgcgtgcaa aacacacaag ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag
      421 acaaatggct ctagagggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
      481 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
      541 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
      601 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
      661 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
      721 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
      781 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
      841 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
      901 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
      961 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     1021 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     1081 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     1141 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     1201 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     1261 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     1321 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     1381 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1441 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1501 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1561 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1621 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1681 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1741 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1801 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1861 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1921 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     1981 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2041 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2101 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2161 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc
     2221 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     2281 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2341 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2401 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2461 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2521 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2581 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2641 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     2701 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2761 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2821 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2881 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2941 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3001 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3061 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3121 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
