LOCUS       Exported                2958 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses gRNA from hybrid human U6/T7 promoter for both cellular 
            expression and in vitro transcription from same promoter (2 Gs at 
            initiation).
ACCESSION   .
VERSION     .
KEYWORDS    pU6T7
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2958)
  AUTHORS   Romanienko PJ, Giacalone J, Ingenito J, Wang Y, Isaka M, Johnson T, 
            You Y, Mark WH
  TITLE     A Vector with a Single Promoter for In Vitro Transcription and 
            Mammalian Cell Expression of CRISPR gRNAs.
  JOURNAL   PLoS One. 2016 Feb 5;11(2):e0148362. doi: 
            10.1371/journal.pone.0148362. eCollection 2016.
  PUBMED    26849369
REFERENCE   2  (bases 1 to 2958)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..2958
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     446..466
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     617..636
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        678..696
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        718..793
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(868..884)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(868..884)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(881..903)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    892..908
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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(916..946)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    961..982
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1099..1116)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1270..1858)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1350..1369)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2036..2845)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2759..2778
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2837..2856
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca
      481 tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat
      541 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
      601 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
      661 ggctttatat atcttgctaa tacgactcac tataggagag accgagagag ggtctcagtt
      721 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      781 accgagtcgg tgcttttttt aaatctagat atcggatccc gggcccgtcg actgcagagg
      841 cctgcatgca agcttggtgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
      901 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
      961 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     1021 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     1081 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     1141 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     1201 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     1261 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     1321 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     1381 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     1441 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     1501 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     1561 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     1621 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     1681 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     1741 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     1801 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     1861 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     1921 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     1981 aagttttaaa tcaagcccaa tctgaataat gttacaacca attaaccaat tctgattaga
     2041 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     2101 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     2161 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     2221 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     2281 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     2341 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     2401 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     2461 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     2521 ctaatacctg gaatgctgtt tttccgggga tcgcagtggt gagtaaccat gcatcatcag
     2581 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     2641 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     2701 ctggcgcatc gggcttccca tacaagcgat agattgtcgc acctgattgc ccgacattat
     2761 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg
     2821 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     2881 gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag
     2941 acacgggcca gagctgca
//
