LOCUS       sequence-128761-        2957 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses gRNA from hybrid human U6/T7 promoter for both cellular 
            expression and in vitro transcription from same promoter (1 G at 
            initiation).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2957)
  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 2957)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2016-02-5"; volume: "11(2):e0148362. doi"; pages: " 
            10.1371/journal.pone.0148362. eCollection 2016"
COMMENT     Sequence Label: pU6T7G
FEATURES             Location/Qualifiers
     source          1..2957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     primer_bind     678..697
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        678..696
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        717..792
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(867..883)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(867..883)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(880..902)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    891..907
                     /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(join(915..921,922..939,940..945))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    960..981
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1098..1115)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1269..1857)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1349..1368)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2035..2844)
                     /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     2758..2777
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2836..2855
                     /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 tatagggaga ccgagagagg gtctcagttt
      721 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
      781 ccgagtcggt gcttttttta aatctagata tcggatcccg ggcccgtcga ctgcagaggc
      841 ctgcatgcaa gcttggtgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
      901 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
      961 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     1021 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     1081 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     1141 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     1201 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     1261 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     1321 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     1381 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     1441 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     1501 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     1561 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     1621 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     1681 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     1741 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     1801 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     1861 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     1921 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     1981 agttttaaat caagcccaat ctgaataatg ttacaaccaa ttaaccaatt ctgattagaa
     2041 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     2101 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     2161 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     2221 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     2281 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     2341 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     2401 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     2461 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     2521 taatacctgg aatgctgttt ttccggggat cgcagtggtg agtaaccatg catcatcagg
     2581 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     2641 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     2701 tggcgcatcg ggcttcccat acaagcgata gattgtcgca cctgattgcc cgacattatc
     2761 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga
     2821 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     2881 ttttattgtt catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga
     2941 cacgggccag agctgca
//
