LOCUS       sequence-244225-        2743 bp    DNA     circular SYN 15-APR-2025
DEFINITION  expression of gRNA targeting HAS2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2743)
  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 2743)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: gh176
FEATURES             Location/Qualifiers
     source          1..2743
                     /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        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(485..502)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(595..1183)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(675..694)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1354..2145,2146..2214))
                     /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     1977..1996
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2215..2319)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2620..2642
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2634..2651
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2635..2651
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2662..2681
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2662..2680
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
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 tgaaaaggct aacctaccct gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttggtacct
      361 ggagcacaag actggcctca tgggccttcc gctcactgcc cgctttccag tcgggaaacc
      421 tgtcgtgcca gctgcattaa catggtcata gctgtttcct tgcgtattgg gcgctctccg
      481 cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt aaagcctggg gtgcctaatg
      541 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
      601 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
      661 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
      721 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
      781 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
      841 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
      901 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
      961 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     1021 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     1081 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     1141 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     1201 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     1261 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     1321 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     1381 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     1441 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaacc
     1501 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     1561 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     1621 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     1681 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     1741 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     1801 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     1861 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     1921 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     1981 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     2041 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     2101 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     2161 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     2221 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     2281 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     2341 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     2401 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     2461 gagatagggt tgagtggccg ctacagggcg ctcccattcg ccattcaggc tgcgcaactg
     2521 ttgggaaggg cgtttcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     2581 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     2641 cgacggccag tgagcgcgac gtaatacgac tcactatagg gcgaattggc ggaaggccgt
     2701 caaggccacg tgtcttgtcc agagctcgat atcgaattaa ttc
//
