LOCUS       40924_24034        3564 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses the S. pyogenes sgRNA from the 7SK promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3564)
  AUTHORS   Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA
  TITLE     Multiplex CRISPR/Cas9-based genome engineering from a single 
            lentiviral vector.
  JOURNAL   Nucleic Acids Res. 2014 Aug 13. pii: gku749.
  PUBMED    25122746
REFERENCE   2  (bases 1 to 3564)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3564)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2014 Aug 13. pii: gku749."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3564
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        327..402
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(458..476)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(497..513)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(521..537)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(545..575)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(590..611)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(728..745)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(899..1487)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1627..2418)
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(2453..2810)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        complement(2812..2916)
                     /label=AmpR promoter
     rep_origin      complement(2942..3397)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     3539..3555
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg cgaattgggt acctttaatt ctagtactat gcatcgtctc
       61 attgtctgca gtatttagca tgccccaccc atctgcaagg cattctggat agtgtcaaaa
      121 cagccggaaa tcaagtccgt ttatctcaaa ctttagcatt ttgggaataa atgatatttg
      181 ctatgctggt taaattagat tttagttaaa tttcctgctg aagctctagt acgataagca
      241 acttgaccta agtgtaaagt tgagacttcc ttcaggttta tatagcttgt gcgccgcttg
      301 ggtacctcgg gtcttcgaga agacctgttt tagagctaga aatagcaagt taaaataagg
      361 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc tcggactgat
      421 gacctgagac gccgcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc
      481 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
      541 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
      601 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
      661 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
      721 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
      781 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
      841 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
      901 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
      961 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     1021 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     1081 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     1141 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     1201 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     1261 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     1321 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     1381 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     1441 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     1501 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     1561 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcatca
     1621 tgatcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc
     1681 gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc
     1741 acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat
     1801 gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt
     1861 cacgacgaga tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca gttcggctgg
     1921 cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg
     1981 agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc
     2041 aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag
     2101 gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc
     2161 ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag
     2221 ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag
     2281 aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg
     2341 ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa
     2401 tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcga tctttgcaaa
     2461 agcctaggcc ttcaaaaaag cctcctcact acttctggaa tagctcagag gccgaggcgg
     2521 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac
     2581 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat
     2641 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg
     2701 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
     2761 ttccacaccc taactgacac acattccaca gctggttctt tccgcctcag gactcttcct
     2821 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     2881 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     2941 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     3001 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     3061 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     3121 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     3181 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     3241 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     3301 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     3361 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
     3421 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     3481 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     3541 aaaacgacgg ccagtgagcg cgcg
//
