LOCUS       sequence-418944-        5910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mutant AlkB protein (D135S) in E. coli BL21(DE3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5910)
  AUTHORS   Zheng G, Qin Y, Clark WC, Dai Q, Yi C, He C, Lambowitz AM, Pan T
  TITLE     Efficient and quantitative high-throughput tRNA sequencing.
  JOURNAL   Nat Methods. 2015 Sep;12(9):835-7. doi: 10.1038/nmeth.3478. Epub 
            2015 Jul 27.
  PUBMED    26214130
REFERENCE   2  (bases 1 to 5910)
  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: 
            "10.1038/nmeth.3478"; journalName: "Nat Methods"; date: "2015-09"; 
            volume: "12"; issue: "9"; pages: "835-7"
COMMENT     Sequence Label: pET30a-AlkB-D135S
FEATURES             Location/Qualifiers
     source          1..5910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(54..101)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     97..115
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(168..185)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(816..833)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(843..860)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             879..901
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    916..940
                     /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(940..959)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(941..959)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1043..1062)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1268..1345
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1346..2428
                     /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(1365..1384)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3237..3428
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3422..3444)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3530..3669
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3585..3604
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3684..3701)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3855..4443)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3935..3954)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4554..4573)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4565..5380
                     /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             4565..5380
                     /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(4638..4657)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5472..5910,1..17))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5609..5630)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5821..5840
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccgctacag ggcgcgtccc attcgccaat ccggatatag ttcctccttt cagcaaaaaa
       61 cccctcaaga cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca
      121 gcagccaact cagcttcctt tcgggctttg ttagcagccg gatctcagtg gtggtggtgg
      181 tggtgctcga gttattcttt tttacctgcc tgacggaatg tcaggttgta gcggcagtcg
      241 atggtgagtg gatgaaaccc cgctttcaac ggttgaatac cgtgataaaa cagccgcgat
      301 tcaccgcccc ataccaccac atcgccatgt tccaacaaca aacgtttgag cggatcatta
      361 cgtttcaggc cgccaaattg aaaaatcgcg ggtaagccca gagaaacaga aacaattggc
      421 gcgcgcagat ccggttcaga tttatcctga tgcagcgaca gtttcgcgcc aggagcgtag
      481 cggttgataa gacaagcatc tggctggaaa tctggatagc ccgccgccgt agccgcacgt
      541 tgacataaat tatgaaaact ctgtggcatg gcgggccacg gtttatttgt ttgcggatca
      601 atgggcgaat agagataacc ttgccgatgg gtcgtccagc ccagatgccc acagttggtc
      661 atcgccaccg acatggtata tcccccgggg gtgaccatct ggcgaaacgg cgactggctg
      721 gcaacgtcat taatatcgcg gattagttgc tccgcagcgt taaaagcaaa acgccgtaaa
      781 attaccgctc cagccgccag tggctcttgc atatggctgc cgcgcggcac caggccgctg
      841 ctgtgatgat gatgatgatg gctgctgccc atggtatatc tccttcttaa agttaaacaa
      901 aattatttct agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa
      961 tttcgcggga tcgagatctc gatcctctac gccggacgca tcgtggccgg catcaccggc
     1021 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct
     1081 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc
     1141 gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac
     1201 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga
     1261 gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga
     1321 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     1381 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     1441 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     1501 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     1561 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     1621 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     1681 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     1741 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     1801 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     1861 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     1921 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     1981 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     2041 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     2101 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
     2161 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     2221 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     2281 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     2341 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2401 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     2461 attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc
     2521 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac
     2581 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga
     2641 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag
     2701 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca
     2761 tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc
     2821 ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact
     2881 gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt
     2941 cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg
     3001 caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt
     3061 gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac
     3121 aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc
     3181 gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga
     3241 ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc
     3301 ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc
     3361 acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa
     3421 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     3481 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     3541 cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat
     3601 tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     3661 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3721 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3781 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3841 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3901 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3961 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4021 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4081 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4141 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4201 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4261 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     4321 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4381 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4441 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4501 taagggattt tggtcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     4561 tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat
     4621 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     4681 aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     4741 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     4801 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     4861 tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     4921 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     4981 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     5041 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     5101 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     5161 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     5221 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     5281 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     5341 taaattgcag tttcatttga tgctcgatga gtttttctaa gaattaattc atgagcggat
     5401 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5461 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     5521 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     5581 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     5641 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     5701 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     5761 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     5821 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     5881 cgtaaccacc acacccgccg cgcttaatgc
//
