LOCUS       40924_48788        3008 bp DNA     circular SYN 13-MAY-2021
DEFINITION  sgRNA cloning backbone with MS2 loops at tetraloop and stemloop 2. 
            Contains BbsI sites for insertion of spacer sequences..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3008)
  AUTHORS   Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena 
            C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F
  TITLE     Genome-scale transcriptional activation by an engineered CRISPR-Cas9
            complex.
  JOURNAL   Nature. 2014 Dec 10. doi: 10.1038/nature14136.
  PUBMED    25494202
REFERENCE   2  (bases 1 to 3008)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3008)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2014 Dec 10. doi: 10.1038/nature14136."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..247
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        291..309
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat
                     protein"
     misc_RNA        361..379
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat
                     protein"
     rep_origin      491..946
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(963..982)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     1082..1104
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1142..1160)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        1228..1332
                     /label=AmpR promoter
     CDS             1333..2190
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2364..2952
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
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 acaccgggtc ttcgagaaga cctgttttag agctaggcca acatgaggat
      301 cacccatgtc tgcagggcct agcaagttaa aataaggcta gtccgttatc aacttggcca
      361 acatgaggat cacccatgtc tgcagggcca agtggcaccg agtcggtgct ttttttgaat
      421 tctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
      481 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
      541 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
      601 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
      661 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
      721 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
      781 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
      841 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
      901 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
      961 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     1021 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     1081 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     1141 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     1201 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     1261 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     1321 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     1381 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     1441 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     1501 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     1561 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     1621 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     1681 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     1741 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     1801 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     1861 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     1921 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     1981 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     2041 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     2101 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     2161 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     2221 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     2281 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2341 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2401 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2461 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     2521 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2581 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2641 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2701 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2761 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     2821 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     2881 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     2941 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3001 tttgctca
//
