LOCUS       sequence-146703-        3010 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sgRNA2.0 targeting 138nt upstream of TSS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3010)
  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 3010)
  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_SAM2
FEATURES             Location/Qualifiers
     source          1..3010
                     /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        293..311
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        363..381
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     rep_origin      493..948
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(580..599)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     790..811
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(965..984)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1084..1106
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1144..1162)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1230..1334
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1335..1403,1404..2195)
                     /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(1553..1572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2366..2954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2855..2874
                     /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 acaccgggag tgctgacact ggggggtttt agagctaggc caacatgagg
      301 atcacccatg tctgcagggc ctagcaagtt aaaataaggc tagtccgtta tcaacttggc
      361 caacatgagg atcacccatg tctgcagggc caagtggcac cgagtcggtg ctttttttga
      421 attctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
      481 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
      541 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
      601 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      661 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
      721 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
      781 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
      841 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
      901 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
      961 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     1021 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     1081 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     1141 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     1201 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1261 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1321 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1381 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1441 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1501 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1561 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1621 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1681 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1741 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1801 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1861 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1921 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1981 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2041 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2101 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2161 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2221 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2281 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2341 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2401 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2461 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2521 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2581 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2641 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2701 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2761 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2821 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2881 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2941 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3001 cttttgctca
//
