LOCUS       sequence-459421-        5980 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express wild type AlkB protein in E. coli BL21(DE3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5980)
  AUTHORS   Shi J, Zhang Y, Tan D, Zhang X, Yan M, Zhang Y, Franklin R, Shahbazi
            M, Mackinlay K, Liu S, Kuhle B, James ER, Zhang L, Qu Y, Zhai Q, 
            Zhao W, Zhao L, Zhou C, Gu W, Murn J, Guo J, Carrell DT, Wang Y, 
            Chen X, Cairns BR, Yang XL, Schimmel P, Zernicka-Goetz M, Cheloufi 
            S, Zhang Y, Zhou T, Chen Q
  TITLE     PANDORA-seq expands the repertoire of regulatory small RNAs by 
            overcoming RNA modifications.
  JOURNAL   Nat Cell Biol. 2021 Apr;23(4):424-436. doi: 
            10.1038/s41556-021-00652-7. Epub 2021 Apr 5.
  PUBMED    33820973
REFERENCE   2  (bases 1 to 5980)
  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/s41556-021-00652-7"; journalName: "Nat Cell Biol"; date: 
            "2021-04"; volume: "23"; issue: "4"; pages: "424-436"
COMMENT     Sequence Label: pET28a-AlkB
FEATURES             Location/Qualifiers
     source          1..5980
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(858..875)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(885..902)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             921..943
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    958..982
                     /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(982..1001)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(983..1001)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1085..1104)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1310..1387
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1388..2470
                     /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(1407..1426)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3279..3470
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3464..3486)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3572..3711
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3627..3646
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3726..3743)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3897..4485)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3977..3996)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4596..4615)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4607..5422
                     /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             4607..5422
                     /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(4680..4699)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5514..5969)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5651..5672)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5863..5882
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccttattct ttcttacccg cttgacggaa ggtcaggtta
      241 taacggcagt caatggtcag cgggtgaaag cccgctttca gcggctggat accgtggtag
      301 aacagacggc tttcgccacc ccacacaacc acatcgccgt gctccagcag cagacgtttc
      361 agcgggtcgt tacgcttcag gccaccaaac tggaaaatcg ccggcagacc caggctcacg
      421 ctaacgatcg gcgcacgcag atccggctca tctttgtctt ggtgcaggct cagcttcgca
      481 cccggcgcat aacggttaat caggcacgca tccggctgaa agtccgggta acccgccgcg
      541 gtcgccgcac gttggcacag gttgtggaag ctctgcggca tcgccggcca cggcttgttg
      601 gtttgcgggt cgatcgggct atacaggtaa ccctgacggt gggtggtcca acccaggtga
      661 ccgcagttgg tcatcgccac gctcatggtg tagccacccg gggtaaccat ttgacgaaac
      721 gggctctggc tcgccacatc gttaatgtca cggatcagtt gctccgccgc gttaaacgcg
      781 aaacgacgca ggataaccgc acccgccgcc agcggttcct gccacggctc cgcatccgcg
      841 aacaggtcca gcatatggct gccgcgcggc accaggccgc tgctgtgatg atgatgatga
      901 tggctgctgc ccatggtata tctccttctt aaagttaaac aaaattattt ctagagggga
      961 attgttatcc gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc
     1021 tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct
     1081 ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg
     1141 agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc
     1201 atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg
     1261 ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga
     1321 atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
     1381 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
     1441 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
     1501 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
     1561 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
     1621 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
     1681 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
     1741 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
     1801 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
     1861 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
     1921 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
     1981 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
     2041 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
     2101 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
     2161 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
     2221 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     2281 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     2341 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
     2401 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     2461 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc
     2521 gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga
     2581 ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg
     2641 cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc
     2701 tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg
     2761 ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc
     2821 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt
     2881 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg
     2941 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc
     3001 ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac
     3061 cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc
     3121 tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg
     3181 catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta
     3241 cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg
     3301 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg
     3361 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc
     3421 tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     3481 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     3541 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     3601 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     3661 ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     3721 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3781 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3841 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3901 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3961 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4021 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4081 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4141 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4201 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4261 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4321 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4381 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4441 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4501 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4561 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     4621 acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg
     4681 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
     4741 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     4801 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     4861 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac
     4921 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     4981 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     5041 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     5101 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     5161 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     5221 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     5281 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     5341 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     5401 gatgctcgat gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt
     5461 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     5521 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     5581 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     5641 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     5701 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     5761 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     5821 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     5881 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     5941 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
