LOCUS       sequence-146702-        3011 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sgRNA2.0 targeting 158nt upstream of TSS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3011)
  AUTHORS   Kaczmarczyk L, Mende Y, Zevnik B, Jackson WS
  TITLE     Manipulating the Prion Protein Gene Sequence and Expression Levels 
            with CRISPR/Cas9.
  JOURNAL   PLoS One. 2016 Apr 29;11(4):e0154604. doi: 
            10.1371/journal.pone.0154604. eCollection 2016.
  PUBMED    27128441
REFERENCE   2  (bases 1 to 3011)
  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: "PLoS One.";
            date: "2016-04-29"; volume: "11(4):e0154604. doi"; pages: " 
            10.1371/journal.pone.0154604. eCollection 2016"
COMMENT     Sequence Label: sgRNA(MS2)_Prnp_SAM1
FEATURES             Location/Qualifiers
     source          1..3011
                     /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        294..312
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        364..382
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      494..949
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(581..600)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     791..812
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(966..985)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1085..1107
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1145..1163)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1231..1335
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1336..1404,1405..2196)
                     /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(1554..1573)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2367..2955
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2856..2875
                     /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 acaccgatag ttgctgagcg tcgtcagttt tagagctagg ccaacatgag
      301 gatcacccat gtctgcaggg cctagcaagt taaaataagg ctagtccgtt atcaacttgg
      361 ccaacatgag gatcacccat gtctgcaggg ccaagtggca ccgagtcggt gctttttttg
      421 aattctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
      481 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
      541 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
      601 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
      661 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
      721 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
      781 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
      841 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
      901 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
      961 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     1021 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     1081 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     1141 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     1201 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1261 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     1321 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     1381 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     1441 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     1501 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     1561 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     1621 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     1681 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     1741 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     1801 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     1861 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     1921 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     1981 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2041 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2101 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2161 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     2221 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     2281 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2341 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2401 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2461 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     2521 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2581 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2641 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2701 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2761 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     2821 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     2881 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     2941 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3001 ccttttgctc a
//
