LOCUS       Exported                5918 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Express wild type AlkB protein in E. coli BL21(DE3).
ACCESSION   .
VERSION     .
KEYWORDS    pET30a-AlkB
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5918)
  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 5918)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5918
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      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(788..805)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(815..832)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             851..873
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    888..912
                     /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(912..931)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(913..931)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1019..1038)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1244..1321
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1322..2404
                     /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(1341..1360)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3213..3404
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3398..3420)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3506..3648
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3561..3580
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3663..3680)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3834..4422)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3914..3933)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4533..4552)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4544..5359
                     /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(4617..4636)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5452..5907)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5589..5610)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5801..5820
                     /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 gagttattct tttttacctg
      181 cctgacggaa tgtcaggttg tagcggcagt cgatggtgag tggatgaaac cccgctttca
      241 acggttgaat accgtgataa aacagccgcg attcaccgcc ccataccacc acatcgccat
      301 gttccaacaa caaacgtttg agcggatcat tacgtttcag gccgccaaat tgaaaaatcg
      361 cgggtaagcc cagagaaaca gaaacaattg gcgcgcgcag atccggttcg tctttatcct
      421 gatgcagcga cagtttcgcg ccaggagcgt agcggttgat aagacaagca tctggctgga
      481 aatctggata gcccgccgcc gtagccgcac gttgacataa attatgaaaa ctctgtggca
      541 tggcgggcca cggtttattt gtttgcggat caatgggcga atagagataa ccttgccgat
      601 gggtcgtcca gcccagatgc ccacagttgg tcatcgccac cgacatggta tatcccccgg
      661 gggtgaccat ctggcgaaac ggcgactggc tggcaacgtc attaatatcg cggattagtt
      721 gctccgcagc gttaaaagca aaacgccgta aaattaccgc tccagccgcc agtggctctt
      781 gcatatggct gccgcgcggc accaggccgc tgctgtgatg atgatgatga tggctgctgc
      841 ccatggtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
      901 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc
      961 ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc
     1021 tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct
     1081 tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc
     1141 ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc
     1201 ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg
     1261 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     1321 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     1381 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     1441 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     1501 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     1561 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     1621 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     1681 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     1741 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     1801 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     1861 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     1921 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     1981 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     2041 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     2101 cgggctgcgc gttggtgcgg acatctcggt agtgggatac gacgataccg aagacagctc
     2161 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     2221 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     2281 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     2341 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     2401 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga
     2461 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     2521 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     2581 tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc
     2641 ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca
     2701 aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg gtgcgcatga
     2761 tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga
     2821 atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct
     2881 gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt
     2941 cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg
     3001 gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt
     3061 cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt
     3121 catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca
     3181 tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta
     3241 acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg
     3301 acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc
     3361 gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     3421 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     3481 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     3541 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatatg
     3601 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
     3661 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3721 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3781 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3841 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3901 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3961 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4021 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4081 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4141 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4201 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4261 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4321 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4381 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4441 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaac
     4501 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg
     4561 ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
     4621 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa
     4681 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
     4741 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca
     4801 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc
     4861 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
     4921 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt
     4981 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt
     5041 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt
     5101 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
     5161 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     5221 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     5281 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     5341 gctcgatgag tttttctaag aattaattca tgagcggata catatttgaa tgtatttaga
     5401 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaaattgtaa
     5461 acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     5521 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     5581 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     5641 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     5701 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     5761 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     5821 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     5881 cgcttaatgc gccgctacag ggcgcgtccc attcgcca
//
