LOCUS       sequence-439539-        5986 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Yjjgin E. coli BL21_DE3 (pLys) or similar.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5986)
  TITLE     Efficient synthetic access to stable isotope labelled pseudouridine 
            phosphoramidites for RNA NMR spectroscopy
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5986)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pet28a+_Yjjg
FEATURES             Location/Qualifiers
     source          1..5986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    33..57
                     /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)."
     primer_bind     complement(57..76)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(58..76)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(160..179)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        385..462
                     /gene="lacI"
                     /label=lacI promoter
     CDS             463..1545
                     /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     complement(482..501)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2354..2545
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2539..2561)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2647..2789
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2702..2721
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2804..2821)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2975..3563)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3055..3074)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3674..3693)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3685..4500
                     /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             3685..4500
                     /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     complement(3758..3777)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4593..5048)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4730..4751)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4942..4961
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5085..5132)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5128..5146
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5199..5216)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5919..5936)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(5946..5963)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             join(5982..5986,1..18)
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct
       61 atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc ggacgcatcg
      121 tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg
      181 atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg
      241 tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg
      301 cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc
      361 ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg
      421 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta
      481 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
      541 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
      601 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
      661 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
      721 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
      781 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
      841 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
      901 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
      961 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     1021 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     1081 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     1141 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
     1201 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     1261 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
     1321 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     1381 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     1441 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     1501 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     1561 tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa
     1621 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt
     1681 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga
     1741 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt
     1801 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca
     1861 ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     1921 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     1981 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     2041 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     2101 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     2161 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     2221 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     2281 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     2341 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     2401 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     2461 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
     2521 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     2581 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     2641 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     2701 gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa ataccgcaca
     2761 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     2821 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2881 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2941 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3001 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3061 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3121 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3181 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3241 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3301 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3361 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     3421 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3481 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3541 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3601 agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact gtctgcttac
     3661 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctctagg
     3721 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat
     3781 gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg
     3841 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
     3901 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat
     3961 gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa
     4021 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat
     4081 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg
     4141 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
     4201 tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca
     4261 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
     4321 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     4381 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt
     4441 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
     4501 gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     4561 tccgcgcaca tttccccgaa aagtgccacc tgaaattgta aacgttaata ttttgttaaa
     4621 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     4681 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     4741 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     4801 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     4861 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     4921 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     4981 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     5041 gggcgcgtcc cattcgccaa tccggatata gttcctcctt tcagcaaaaa acccctcaag
     5101 acccgtttag aggccccaag gggttatgct agttattgct cagcggtggc agcagccaac
     5161 tcagcttcct ttcgggcttt gttagcagcc ggatctcagt ggtggtggtg gtggtgctcg
     5221 agtgcggccg caagctttca gtgtttacac aggagctgct ccagttcgtg caacgaagaa
     5281 acggtccagg tgggcgcgat gccttctggt tgctcgcgat ggtgtgcatt cagccagcag
     5341 gtcgcaagcc cggcgttgat gccaccgaga atatcggact cggcagtgtc gccaaccatc
     5401 agcacgcgtg aacggtcagg attgcccgcc tgttccagcg cataatcgaa aattttctta
     5461 ttcggtttgg caacgccaac ttcttcggaa atcaccagca aatcgaagta atcacgcagg
     5521 cccgtgcgtt ccagacgcac ctgttgcaag gcactaaagc cgttggtgat gatgccgatt
     5581 ttggcgttgc cacgaatggc gttaagcaga gaaaccgcgc ccggcagcgg cgtgcagatt
     5641 tccgccatcg cattaataaa ggcttcgttg agtttacctg gctcgacgtt cagccgttcg
     5701 gcccagctct caaaccgccc gtgctgaagc tgtaatgaag tgatcgcgcc gttttgataa
     5761 tccacccaca gtggcttgtt aacggcctga tagtcctgaa aatcttcagc ggtaaaggtg
     5821 acgctgtaat caagaaacat ccgctgcagg ccggtgaatg agtcaaaggt aaacagcgtt
     5881 tcatcggcat caaagaaaat ccagtcccac ttcatatggc tgccgcgcgg caccaggccg
     5941 ctgctgtgat gatgatgatg atggctgctg cccatggtat atctcc
//
