LOCUS       sequence-348166-        7102 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses Legionella pneumophila SidK (1-278) with a cleavable 
            N-terminal His tag (TEV site) and C-terminal 3xFLAG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7102)
  AUTHORS   Abbas YM, Wu D, Bueler SA, Robinson CV, Rubinstein JL
  TITLE     Structure of V-ATPase from the mammalian brain.
  JOURNAL   Science. 2020 Mar 13;367(6483):1240-1246. doi: 
            10.1126/science.aaz2924.
  PUBMED    32165585
REFERENCE   2  (bases 1 to 7102)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2020-03-13- 13"; volume: "367"; 
            issue: "6483"; pages: "1240-1246"
COMMENT     Sequence Label: pSAB35
FEATURES             Location/Qualifiers
     source          1..7102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     220..239
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     473..490
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    505..644
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(570..589)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     730..752
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(746..937)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1746..2828)
                     /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     2790..2809
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2829..2906)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3112..3131
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3215..3234
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3215..3233
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3234..3258
                     /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             3273..3295
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3305..3322
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3323..3343
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4178..4243
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5181..5198
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5251..5269)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5265..5312
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5349..5804
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5436..5455)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5646..5667
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5896..6711)
                     /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(5896..6711)
                     /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     6619..6638
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6703..6722
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(6833..7102,1..319)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
       61 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
      121 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
      181 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
      241 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
      301 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
      361 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
      421 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
      481 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
      541 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
      601 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
      661 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
      721 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
      781 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
      841 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
      901 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
      961 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     1021 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     1081 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     1141 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     1201 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     1261 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     1321 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     1381 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     1441 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     1501 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     1561 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     1621 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     1681 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     1741 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     1801 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     1861 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     1921 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     1981 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     2041 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     2101 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     2161 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     2221 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     2281 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     2341 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     2401 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     2461 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     2521 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     2581 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     2641 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     2701 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     2761 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     2821 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     2881 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     2941 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     3001 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     3061 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     3121 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     3181 ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg
     3241 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     3301 catgcatcat catcatcatc acgaaaactt gtatttccag ggcatgtctt ttatcaaggt
     3361 aggtataaaa atgggtggct taactagcga gcaatatcat agtcaagtag tcggtaaaat
     3421 cggatatatc gccagatgca tgcaaactat agatcctgaa aataatttga aaaaaataag
     3481 ggaagattac caagatgtat tgatatgggc agagaaaaat tatcgttttg aggaaatatt
     3541 ggaagccagc aaatctggaa aatgccctaa tgatctggat gcattaagtc gacgatcgct
     3601 aatactccaa gaattgctgc ggcttgtttc atcaattagt ccctttaaaa tgaaattaga
     3661 cctgattgag tctcaatatg agaaaatgaa gcagcatgtg aatttatgga aaagtgatta
     3721 ccatgtaaaa ctaaatcaac tgaatcaatt aacagattac ctcaaaaatg cagcaccaac
     3781 tcctaaaaat aattttctaa gagcaatgac ctctgtattg cagatgcaga tagcccaata
     3841 tgggattaca gaagataatg aaggcatcaa ccagttgttt aaactgggat tgcatttgct
     3901 cgcaatggct aacgaaaaaa tagatgaaca atatcacctg tttaaagggt atgtaaaaga
     3961 tcagcctgaa gaatctcctt ttgaaggtat tttgcctgcg gaagaccaga aaattttagt
     4021 taaaactatg atagattatg caatgccgaa gctttcaagc aaggttttac aagataaatt
     4081 atcggcacta tccagttctg atgtattaac aaaaacatta cttgatagca tcgacagaat
     4141 tgttaaagaa aacgaaaaat taaatgcttt aagcaaagac tacaaagacc atgacggtga
     4201 ttataaagat catgatatcg attacaagga tgacgatgac aagtgagtca aattaggtaa
     4261 atttggcctg gatatcaggg aaattgaagt gatttactct caagctctga aaatatcacc
     4321 acaagatgct ttgcaatata ctgctcaaca atgtgatgcc caattattaa gcatggcatt
     4381 tcctgattcc caaaattaca ttattgaatc tatatcgaac aaaaaggtca agaccattgc
     4441 agagctaatc catagtaaag aatttattta tcaaattatt aagacagaag tttttaaaca
     4501 agttgatcct aatgagaaaa ttcgactaca agctgcgaca gaattgtatc agcttcttgg
     4561 gagaataatg gataaacaga ttaatctttt taccaagatg aatctcgaac aaattaatga
     4621 atatattcaa accaaaacca aagcaatcct ggacaaaatt cctgaaagag ttgagttatt
     4681 aacttttatg ggctttgaaa ttccaacttt caagggaatt gagacactta tgaccgacat
     4741 ctcccattct caggataatg aaactctggc gatagcccaa gaattttata caaatataaa
     4801 aaatgcaaag aaccaattgt tgggtgataa actgattgaa gacattactc cacaggatgt
     4861 agagaaattt tttaatcaat gtagtcaata cggttcagaa gcagctgaaa agttagctga
     4921 caatcgtccg gtattgacaa aaatagccga tattttgaca gccattgccc gttgggcgat
     4981 tagtttaata ggtttcaata cgccaccgca gtttttggct ccaaccagaa cctgtgtgga
     5041 tcaggtatct gatgaaatca ctaaaattaa actcaaactt gaagatactt tgggaagctt
     5101 gcaaaaagta caggaagaaa gcctaagcct ttaaggatcc gaattcgagc tccgtcgaca
     5161 agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     5221 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     5281 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     5341 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     5401 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     5461 tctcgccacg ttcgccggct ttccccgtca agcactaaat cgggggctcc ctttagggtt
     5521 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     5581 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5641 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5701 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5761 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     5821 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5881 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     5941 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     6001 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     6061 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     6121 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     6181 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     6241 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     6301 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     6361 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     6421 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     6481 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     6541 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     6601 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     6661 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     6721 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     6781 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6841 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6901 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6961 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     7021 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     7081 gcgataagtc gtgtcttacc gg
//
