LOCUS       sequence-372747-        3074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of gRNAs using a human tRNA promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3074)
  AUTHORS   Mishra T, Bhardwaj V, Ahuja N, Gadgil P, Ramdas P, Shukla S, Chande 
            A
  TITLE     Improved loss-of-function CRISPR-Cas9 genome editing in human cells 
            concomitant with inhibition of TGF-beta signaling.
  JOURNAL   Mol Ther Nucleic Acids. 2022 Mar 8;28:202-218. doi: 
            10.1016/j.omtn.2022.03.003. eCollection 2022 Jun 14.
  PUBMED    35402072
REFERENCE   2  (bases 1 to 3074)
  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"; journalName: "Mol Ther 
            Nucleic Acids."; date: "2022-03-8"; volume: "28:202-218. doi"; 
            pages: " 10.1016/j.omtn.2022.03.003. eCollection 2022 Jun 14"
COMMENT     Sequence Label: pTZ tRNA_gRNA empty scaffold
FEATURES             Location/Qualifiers
     source          1..3074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..457
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(89..108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     299..320
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     583..605
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     597..614
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     598..614
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        718..793
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(885..904)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(886..904)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(923..939)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(923..939)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(936..958)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    947..963
                     /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(971..977,978..995,996..1001))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1016..1037
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1154..1171)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1325..1913)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1405..1424)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2084..2875,2876..2944))
                     /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     2707..2726
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2945..3049)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
       61 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
      301 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
      361 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
      421 aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca
      481 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
      541 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      601 aaacgacggc cagtgaattc ggttccatgg tgtaatggtt agcactctgg actctgaatc
      661 cagcgatccg agttcaaatc tcggtggaac ctaaacaccg ggtcttcgag aagacctgtt
      721 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      781 accgagtcgg tgcttttttg ttttagagct agaaatagca agttaaaata aggctagtcc
      841 gtttttagcg cgtgcgccaa ttctgcagac aaatggcaag ctttccctat agtgagtcgt
      901 attagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
      961 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     1021 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     1081 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     1141 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     1201 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     1261 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     1321 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     1381 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     1441 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     1501 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     1561 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     1621 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     1681 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     1741 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     1801 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     1861 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     1921 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     1981 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     2041 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     2101 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     2161 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     2221 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     2281 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     2341 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     2401 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     2461 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     2521 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     2581 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     2641 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     2701 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     2761 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     2821 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     2881 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     2941 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     3001 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     3061 gaaaagtgcc acct
//
