LOCUS       sequence-273191-        4719 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. Coli TGT with a C-terminal His Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4719)
  AUTHORS   Busby KN, Devaraj NK
  TITLE     Enzymatic covalent labeling of RNA with RNA transglycosylation at 
            guanosine (RNA-TAG).
  JOURNAL   Methods Enzymol. 2020;641:373-399. doi: 10.1016/bs.mie.2020.03.009. 
            Epub 2020 Apr 20.
  PUBMED    32713531
REFERENCE   2  (bases 1 to 4719)
  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: "Methods 
            Enzymol."; date: "2020"; volume: "641:373-399. doi"; pages: " 
            10.1016/bs.mie.2020.03.009. Epub 2020 Apr 20"
COMMENT     Sequence Label: TGT-His
FEATURES             Location/Qualifiers
     source          1..4719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(43..64)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     255..274
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     559..581
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     573..590
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     574..590
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     778..797
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        778..796
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             804..826
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1956..1973
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2164..2180)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2164..2180)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2177..2199)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2188..2204
                     /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(2212..2218,2219..2236,2237..2242))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2257..2278
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2395..2412)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2566..3154)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2646..2665)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3750..4559)
                     /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
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDVNKLQFHLMLDEFF"
     primer_bind     4473..4492
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4551..4570
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     4700..4718
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 agcttgggaa accataagac cgagatagag ttgagtgttg ttccagtttg gaacaagagt
       61 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
      121 ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca
      181 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
      241 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctaaggcgct ggcaagtgta
      301 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
      361 tactatggtt gctttgacgt atgcggtgtg aaataccgca cagatgcgta aggagaaaat
      421 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
      481 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
      541 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgatcg
      601 agatcgtgat ccggatcaag atccagatcg aattccatgg tctcaacttt cacaggctgt
      661 cgccgtgctc atttgattga accggtcagc tacggttcga atgcgtcagc gtcagcgatc
      721 tgcagggcca gtgaattcga gctcggtacc tcgcgaatgc atctagatat cggatcctaa
      781 tacgactcac tatagggaat aattttgttt aactttaaga aggagatata atgaaatttg
      841 aactggacac caccgacggt cgcgcacgcc gtggccgcct ggtctttgat cgtggcgtag
      901 tggaaacgcc ttgttttatg cctgttggca cctacggcac cgtaaaaggg atgacgccgg
      961 aagaagttga agccactggc gcgcaaatta tcctcggcaa caccttccac ctgtggctgc
     1021 gcccgggcca ggaaatcatg aaactgcacg gcgatctgca cgattttatg cagtggaagg
     1081 ggccgatcct caccgactcc ggcggcttcc aggtcttcag ccttggcgat attcgtaaaa
     1141 tcaccgaaca gggcgtgcac ttccgtaacc cgatcaacgg cgatccgatt ttcctcgatc
     1201 ctgaaaaatc aatggagatt cagtacgatc ttggttcgga tatcgtcatg atctttgatg
     1261 agtgtacgcc gtatcctgct gactgggatt acgcaaaacg ctccatggag atgtctctgc
     1321 gttgggcgaa gcgtagccgt gagcgttttg acagtctcgg aaacaaaaat gcgctgtttg
     1381 gtatcatcca gggcagcgtt tacgaagatt tacgtgatat ttctgttaaa ggtctggtag
     1441 atatcggttt tgatggctac gctgtcggcg gtctggctgt gggtgagccg aaagcagata
     1501 tgcaccgcat tctggagcat gtatgcccgc aaattccggc agacaaaccg cgttacctga
     1561 tgggcgttgg taaaccagaa gacctggttg aaggcgtacg tcgtggtatc gatatgtttg
     1621 actgcgtaat gccaacccgc aacgcccgaa atggtcattt gttcgtgacc gatggcgtgg
     1681 tgaaaatccg caatgcgaag tataagagcg atactggccc actcgatcct gagtgtgatt
     1741 gctacacctg tcgcaattat tcacgcgctt acttgcatca tcttgaccgt tgcaacgaaa
     1801 tattaggcgc gcgactcaac accattcata accttcgtta ctaccagcgt ttgatggcgg
     1861 gtttacgcaa ggctattgaa gagggtaaat tagagagctt cgtaactgat ttttaccagc
     1921 gtcaggggcg agaagtacca cctttgaacg ttgatcacca tcaccaccat cactaaaaag
     1981 gcgggcctcg agcaaagccc gccgaaaggc gggcttttct gtgtaagctt atcggcagct
     2041 acggtaagct aaggtcgtca gcatcagaag ggatcttgct gccgcccgaa aggagatagg
     2101 atccaagctt gatccagatc ccgatctgga tccagatccg gatcgcagct tggcgtaatc
     2161 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     2221 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     2281 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     2341 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     2401 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2461 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2521 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2581 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2641 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2701 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2761 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     2821 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2881 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     2941 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3001 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3061 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3121 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3181 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3241 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3301 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3361 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3421 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcagcttg ggaaaccata
     3481 agagctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3541 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3601 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3661 acgttaaggg attttggtca tgagcttgcg ccgtcccgtc aagtcagcgt aatgctctgc
     3721 cagtgttaca accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac
     3781 tgcaatttat tcacatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     3841 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     3901 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     3961 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     4021 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     4081 tcaaccaaac cgttattcat tcgtgattgc gcctgagcaa gacgaaatac gcgatcgctg
     4141 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     4201 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgtttttccg
     4261 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     4321 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     4381 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaag
     4441 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     4501 tcagcatcca tgttggaatt taatcgcggc ctcgacgttt cccgttgaat atggctcata
     4561 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt
     4621 ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttc cccccccccc
     4681 catgacatta acctataaaa ataggcgtat cacgaggcc
//
