LOCUS       sequence-217052-        7163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminally His-tagged human CE/RNGTT in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7163)
  AUTHORS   Trotman JB, Giltmier AJ, Mukherjee C, Schoenberg DR
  TITLE     RNA guanine-7 methyltransferase catalyzes the methylation of 
            cytoplasmically recapped RNAs.
  JOURNAL   Nucleic Acids Res. 2017 Oct 13;45(18):10726-10739. doi: 
            10.1093/nar/gkx801.
  PUBMED    28981715
REFERENCE   2  (bases 1 to 7163)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2017-10-13- 13"; volume: "45"; issue: 
            "18"; pages: "10726-10739"
COMMENT     Sequence Label: pET28b-His-hCE
FEATURES             Location/Qualifiers
     source          1..7163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2028..2045
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2098..2116)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2112..2159
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2196..2651
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2283..2302)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2493..2514
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2743..3558)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2743..3558)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3466..3485
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3550..3569
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3680..4268
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4169..4188
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4422..4439
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4454..4593
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4519..4538)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4679..4701
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4695..4886)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5695..6777)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6739..6758
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6778..6855)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7061..7080
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gctagcgctc acaacaagat cccgccgcgg
      181 tggctgaact gtccccggcg cggccagccg gtggcaggaa gattcttacc tctgaagaca
      241 atgttaggac caagatatga tagtcaagtt gctgaagaaa atcggttcca tcccagcatg
      301 ctctcaaatt acctaaagag cctaaaggtt aaaatgggct tgttggtgga cctgacaaat
      361 acttcaaggt tctatgaccg aaatgacata gaaaaagaag gaatcaaata tataaaactt
      421 cagtgtaaag gacatggtga gtgccctacc actgagaata ctgagacctt tattcgtctg
      481 tgtgagcggt ttaatgaaag aaatccacct gaacttatag gtgttcattg tactcatggc
      541 ttcaatcgca ctggtttcct catatgtgcc tttttggtgg agaaaatgga ttggagtatc
      601 gaagcagcag ttgctacttt tgcccaagcc agaccaccag gaatctacaa gggtgattat
      661 ttgaaggaac tttttcgtcg gtatggtgac atagaggaag caccaccccc acctctattg
      721 ccagattggt gttttgagga tgatgaagac gaagatgagg atgaggatgg aaagaaggaa
      781 tcagaacccg ggtcaagtgc ttcttttggc aaaaggagaa aagaacggtt aaaactgggc
      841 gctattttct tggaaggtgt tactgttaaa ggtgtaactc aagtaacaac acaaccaaag
      901 ttaggagagg tacagcagaa gtgtcatcaa ttctgtggct gggaagggtc tggattccct
      961 ggagcacagc ctgtttccat ggacaagcaa aatattaaac ttttagacct gaagccatac
     1021 aaagtaagct ggaaagcaga tggtactcgg tacatgatgt tgattgatgg cacaaatgaa
     1081 gtttttatga ttgatagaga caattcagta tttcatgttt caaatctgga atttccattt
     1141 cgtaaagatc ttcgtatgca tttatcaaat actctcttgg atggcgagat gattattgac
     1201 agagtaaatg gacaggctgt tcctagatat ttgatatatg acataattaa attcaattca
     1261 cagcccgttg gagattgtga ttttaatgtt cgtctgcagt gtatagaacg agaaattata
     1321 agtcctcgac acgaaaaaat gaagactggg ctcattgaca aaacacagga accatttagc
     1381 gtcagaaata agccgttttt tgacatctgt acttcaagaa agctacttga aggaaatttt
     1441 gccaaagaag tgagccatga aatggatgga cttatttttc agcctactgg aaaatacaaa
     1501 cctggtcgat gtgatgatat tttgaaatgg aagcctccca gtctgaattc tgtggatttt
     1561 cgtctaaaaa taacaagaat gggaggagaa gggttacttc ctcagaatgt tggcctcctg
     1621 tatgttggag gttatgaaag accctttgca caaatcaagg tgacaaaaga gctgaaacag
     1681 tatgacaaca aaattataga atgcaaattt gagaacaaca gctgggtctt catgagacag
     1741 agaacagaca aaagttttcc taatgcctac aacactgcca tggctgtgtg taacagcatc
     1801 tcaaaccctg tcaccaagga gatgctgttt gagttcatcg acagatgtac tgcagcttct
     1861 caaggacaga agcgaaaaca tcatctggac cctgacacgg agctcatgcc accaccacct
     1921 cccaaaagac cacacccttt aacctaataa ctagcatgac tggtggacag caaatgggtc
     1981 gggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc
     2041 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     2101 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     2161 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     2221 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2281 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2341 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2401 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2461 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2521 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2581 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2641 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2701 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     2761 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     2821 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     2881 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     2941 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     3001 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     3061 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     3121 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     3181 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     3241 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     3301 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     3361 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     3421 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     3481 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     3541 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     3601 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3661 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3721 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3781 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     3841 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3901 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3961 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4021 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4081 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4141 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4201 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4261 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4321 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4381 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4441 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4501 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4561 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4621 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4681 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     4741 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     4801 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     4861 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     4921 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     4981 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     5041 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     5101 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     5161 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     5221 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     5281 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     5341 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     5401 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     5461 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     5521 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     5581 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     5641 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     5701 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5761 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     5821 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     5881 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     5941 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     6001 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     6061 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     6121 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     6181 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     6241 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     6301 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     6361 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     6421 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     6481 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     6541 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     6601 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     6661 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     6721 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     6781 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     6841 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     6901 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     6961 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     7021 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     7081 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     7141 tcgagatctc gatcccgcga aat
//
