LOCUS       sequence-448063-        5980 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vipp1_dL10Ala with C-terminal 6x His-Tag and
            3C cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5980)
  AUTHORS   Junglas B, Kartte D, Kutzner M, Hellmann N, Ritter I, Schneider D, 
            Sachse C
  TITLE     Structural basis for Vipp1 membrane binding: from loose coats and 
            carpets to ring and rod assemblies.
  JOURNAL   Nat Struct Mol Biol. 2025 Mar;32(3):555-570. doi: 
            10.1038/s41594-024-01399-z. Epub 2024 Oct 8.
  PUBMED    39379528
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/s41594-024-01399-z"; journalName: "Nat Struct Mol Biol"; 
            date: "2025-03"; volume: "32"; issue: "3"; pages: "555-570"
COMMENT     Sequence Label: pET50delCT_Vipp1_dL10Ala
FEATURES             Location/Qualifiers
     source          1..5980
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(149..168)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             160..975
                     /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             160..975
                     /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(233..252)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(1067..1522)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1204..1225)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1416..1435
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1559..1606)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1602..1620
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1701..1745)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(1746..1763)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1837..1854)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1855..1878)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             2687..2709
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2724..2748
                     /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(2748..2767)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2749..2767)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2855..2874)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3080..3157
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3158..4240
                     /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
                     NYIPNRVAQQLAGKQSLLIGVTTSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3177..3196)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4812..5003
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4997..5019)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5105..5244
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5160..5179
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5259..5276)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5430..5980,1..38))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5510..5529)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
       61 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata
      121 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
      181 acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
      241 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
      301 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
      361 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
      421 atccgtactc ctgatgatgc atggttactc accactgcga tccccggcaa aacagcattc
      481 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
      541 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagtga tcgcgtattt
      601 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
      661 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
      721 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
      781 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
      841 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
      901 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
      961 gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa
     1021 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     1081 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     1141 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     1201 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     1261 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     1321 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     1381 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     1441 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     1501 aatgcgccgc tacagggcgc gtcccattcg ccaatccgga tatagttcct cctttcagca
     1561 aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat tgctcagcgg
     1621 tggcagcagc caactcagct tcctttcggg ctttgtttag cagcctaggt taattaagcc
     1681 tcgagagcag cagaagtaga gctgtccatg tgctggcgtt cgaatttagc agcagcggtt
     1741 tctttactac cgcgtggcac cagagcgagc tctgcggccg caagcttgtc gacggacgtc
     1801 gggcgcgcca aggcctgtac agaattcgga tccttaatgg tgatggtggt gatggggtcc
     1861 ctgaaagagg acttcaagag ccgcatttaa ccgacgacgt aggtcgtcca attcctggtc
     1921 aatcaccgca tcatcttgac tggcattatt agctggtact gaggaatcaa caggagccgc
     1981 ttcgagttgg ggcgtagcgg cagaggttcc cggtaatgct ccaccggcca tggaagcttt
     2041 cagggcggcc aactcgtctt ctaccccact gctggcttcc aactgggcaa actggttctc
     2101 gatgccaaag ccggctaact cccctgccgc ttgggaagtg gcttccatat ccagtacctt
     2161 gttctccatc cgttcaaaag ccgctgcagc cgccgctgca gctgctgccg cgagggtttg
     2221 ctgcagttca gcattagcct tggccgcttt ggccctggcc tgcaacatat ttttcttggt
     2281 tttagcttcg gaaatttttg cttctagagc cgcgaggttg cgacggagat tctccgacat
     2341 ggtcctctgt tgggctagct gggtttggta cgccgccgcc gtatctgtca gacttttttt
     2401 gcgggccagg gcttccctag ccaaattttc ttccccattg gtgagggcta attttgcccg
     2461 gtcttcccat ttcttggctt cctgggtgtc ttggttaaga cgttgctctg ttcgtttttc
     2521 ttcggcgatc gtgcgggcaa cggcctggcg gagttgcacc aggtcttcct gcatatcgat
     2581 cacggcctgt tccagaactt tttctggatc ttcagcttta ctgacgaggt cgttgaggtt
     2641 agcccggacg acgcggccta aacggtcaaa taatcccata tgtatatctc cttcttaaag
     2701 ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt
     2761 cgtattaatt tcgcgggatc gagatcgatc tcgatcctct acgccggacg catcgtggcc
     2821 ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg
     2881 gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca
     2941 ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg
     3001 gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag
     3061 ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg
     3121 atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga
     3181 tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtaa accaggccag
     3241 ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat
     3301 tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttaccac
     3361 ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga
     3421 tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa
     3481 agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct
     3541 ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct
     3601 tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg
     3661 actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc
     3721 attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa
     3781 tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca
     3841 aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca
     3901 gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggacat
     3961 ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac
     4021 catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc
     4081 tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac
     4141 caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     4201 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa
     4261 gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag
     4321 tcagctcctt ccggtgggcg cggggcatga ctagcatgat cgtgctcctg tcgttgagga
     4381 cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc
     4441 gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct
     4501 tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt
     4561 tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa
     4621 cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca
     4681 gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg
     4741 tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca
     4801 cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa
     4861 gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca
     4921 tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg
     4981 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     5041 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     5101 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     5161 ggcatcagag cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc
     5221 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5281 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5341 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5401 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5461 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5521 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5581 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5641 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5701 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5761 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5821 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     5881 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5941 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
//
