LOCUS       sequence-223326-        3192 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCR template to construct polycistronic tRNA-gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3192)
  AUTHORS   Xie K, Minkenberg B, Yang Y
  TITLE     Boosting CRISPR/Cas9 multiplex editing capability with the 
            endogenous tRNA-processing system.
  JOURNAL   Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3570-5. doi: 
            10.1073/pnas.1420294112. Epub 2015 Mar 2.
  PUBMED    25733849
REFERENCE   2  (bases 1 to 3192)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1073/pnas.1420294112"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2015-03-17- 17"; volume: "112"; issue: "11"; pages: "3570-5"
COMMENT     Sequence Label: pGTR
FEATURES             Location/Qualifiers
     source          1..3192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      33..42
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      37..46
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        complement(145..220)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(317..335)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(318..335)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(353..369)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(353..369)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(366..388)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    377..393
                     /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(401..407,408..425,426..431))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    446..467
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(584..601)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(755..1343)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(835..854)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1514..2305,2306..2374))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2137..2156
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2375..2479)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2557..3012)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2694..2715)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2906..2925
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3138..3160
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3152..3169
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3153..3169
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(3176..3192,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(3176..3192,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat
       61 tctgccatgc accagccggg aatcgaaccc gggtctgtac cgtggcaggg tactattcta
      121 ccactagacc actggtgctt tgttgcaccg actcggtgcc actttttcaa gttgataacg
      181 gactagcctt attttaactt gctatttcta gctctaaaac cgagacctcg gtctccaatc
      241 actagtgaat tcgcggccgc ctgcaggtcg accatatggg agagctccca acgcgttgga
      301 tgcatagctt gagtattcta tagtgtcacc taaatagctt ggcgtaatca tggtcatagc
      361 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
      421 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
      481 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
      541 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
      601 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
      661 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
      721 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
      781 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
      841 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
      901 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
      961 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     1021 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     1081 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     1141 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     1201 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     1261 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     1321 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     1381 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     1441 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     1501 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     1561 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     1621 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     1681 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     1741 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     1801 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     1861 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     1921 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     1981 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     2041 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     2101 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     2161 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     2221 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     2281 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     2341 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     2401 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     2461 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata
     2521 ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt aatattttgt
     2581 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     2641 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     2701 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     2761 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     2821 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     2881 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     2941 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     3001 tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     3061 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     3121 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata
     3181 cgactcacta ta
//
