LOCUS       40924_48987        3493 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector U6p-gRNA1, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3493)
  AUTHORS   Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK.
  TITLE     Multiplexed and programmable regulation of gene networks with an 
            integrated RNA and CRISPR/Cas toolkit in human cells
  JOURNAL   Mol. Cell 54 (4), 698-710 (2014)
  PUBMED    24837679
REFERENCE   2  (bases 1 to 3493)
  AUTHORS   Perli SD.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-MAY-2014) Electrical Engineering and Computer Science,
            Massachusetts Institute of Technology, 77, Massachusetts Avenue, 
            Cambridge, MA 02139, USA
REFERENCE   3  (bases 1 to 3493)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 3493)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol. Cell";
            date: "2014"; volume: "54"; issue: "4"; pages: "698-710"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (04-MAY-2014) Electrical Engineering and Computer Science, 
            Massachusetts Institute of Technology, 77, Massachusetts Avenue, 
            Cambridge, MA 02139, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3493
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      33..42
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      37..46
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    61..85
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      90..95
                     /label=PolIII terminator
                     /note="PolIII terminator"
                     /regulatory_class="terminator"
     misc_RNA        complement(97..172)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(201..441)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     promoter        complement(618..636)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(654..670)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(678..694)
                     /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(702..732)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(747..768)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(1056..1644)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1818..2675)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(2676..2780)
                     /label=AmpR promoter
     rep_origin      complement(2858..3313)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     3454..3470
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3477..3493
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat
       61 tcaactttgt acaagaaagc tgggtctaga aaaaaagcac cgactcggtg ccactttttc
      121 aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa actgcttcgc
      181 tacacgcgac tcggtgtttc gtcctttcca caagatatat aaagccaaga aatcgaaata
      241 ctttcaagtt acggtaagca tatgatagtc cattttaaaa cataatttta aaactgcaaa
      301 ctacccaaga aattattact ttctacgtca cgtattttgt actaatatct ttgtgtttac
      361 agtcaaatta attctaatta tctctctaac agccttgtat cgtatatgca aatatgaagg
      421 aatcatggga aataggccct cttcctgccc gaccttggta ccggatccag tcgactgaat
      481 tggttccttt aaagcctgct tttttgtaca aggtggcact tttcggggaa atgtgcgaat
      541 cactagtgaa ttcgcggccg cctgcaggtc gaccatatgg gagagctccc aacgcgttgg
      601 atgcatagct tgagtattct atagtgtcac ctaaatagct tggcgtaatc atggtcatag
      661 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
      721 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
      781 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
      841 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
      901 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
      961 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     1021 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     1081 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     1141 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     1201 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     1261 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     1321 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     1381 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     1441 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     1501 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     1561 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     1621 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     1681 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     1741 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     1801 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     1861 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     1921 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     1981 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     2041 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     2101 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     2161 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     2221 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     2281 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     2341 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     2401 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     2461 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     2521 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     2581 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     2641 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     2701 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     2761 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgatgc ggtgtgaaat
     2821 accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt taatattttg
     2881 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     2941 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     3001 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     3061 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     3121 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     3181 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     3241 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     3301 ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     3361 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     3421 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat
     3481 acgactcact ata
//
