LOCUS       sequence-485245-        5986 bp    DNA     circular SYN 03-SEP-2025
DEFINITION  Bacterial expression of Vipp1 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 5986)
  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 5986)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 3, 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
FEATURES             Location/Qualifiers
     source          1..5986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             74..96
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    111..135
                     /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(135..154)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(136..154)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(242..261)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        467..544
                     /gene="lacI"
                     /label=lacI promoter
     CDS             545..1627
                     /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(564..583)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2199..2390
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2384..2406)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2492..2631
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2547..2566
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2646..2663)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2817..3405)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2897..2916)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3516..3535)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3527..4342
                     /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             3527..4342
                     /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(3600..3619)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4434..4889)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4571..4592)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4783..4802
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4926..4973)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4969..4987
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5068..5112)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(5113..5130)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(5204..5221)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5222..5245)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 agctttactg acgaggtcgt tgaggttagc ccggacgacg cggcctaaac ggtcaaataa
       61 tcccatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
      121 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atcgatctcg
      181 atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc
      241 gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc
      301 gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc
      361 tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc
      421 tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg
      481 gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
      541 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
      601 cgtttcccgc gtggtaaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
      661 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
      721 acagtcgttg ctgattggcg ttaccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
      781 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
      841 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
      901 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
      961 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     1021 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     1081 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     1141 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     1201 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     1261 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     1321 gtccgggctg cgcgttggtg cggacatctc ggtagtggga tacgacgata ccgaagacag
     1381 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     1441 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     1501 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     1561 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     1621 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac
     1681 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta
     1741 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt
     1801 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg
     1861 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc
     1921 ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac
     1981 cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc
     2041 tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg
     2101 catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta
     2161 cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg
     2221 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg
     2281 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc
     2341 tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     2401 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     2461 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     2521 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     2581 ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     2641 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2701 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2761 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2821 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2881 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2941 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3001 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3061 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3121 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3181 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3241 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3301 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3361 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3421 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3481 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     3541 acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg
     3601 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
     3661 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     3721 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     3781 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggcaaaac
     3841 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     3901 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagtgatcg
     3961 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     4021 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     4081 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     4141 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     4201 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     4261 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     4321 gatgctcgat gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt
     4381 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     4441 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     4501 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     4561 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     4621 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     4681 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     4741 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     4801 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     4861 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca atccggatat agttcctcct
     4921 ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc tagttattgc
     4981 tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt tgtttagcag cctaggttaa
     5041 ttaagcctcg agagcagcag aagtagagct gtccatgtgc tggcgttcga atttagcagc
     5101 agcggtttct ttactaccgc gtggcaccag agcgagctct gcggccgcaa gcttgtcgac
     5161 ggacgtcggg cgcgccaagg cctgtacaga attcggatcc ttaatggtga tggtggtgat
     5221 ggggtccctg aaagaggact tcaagagccg ccagattatt taaccgacga cgtaggtcgt
     5281 ccaattcctg gtcaatcacc gcatcatctt gactggcatt attagctggt actgaggaat
     5341 caacaggagc cgcttcgagt tggggcgtag cggcagaggt tcccggtaat gctccaccgg
     5401 ccatggaagc tttcagggcg gccaactcgt cttctacccc actgctggct tccaactggg
     5461 caaactggtt ctcgatgcca aagccggcta actcccctgc cgcttgggaa gtggcttcca
     5521 tatccagtac cttgttctcc atccgttcaa aagcactagt agcactgcta gtacctaagc
     5581 ccccgagggt ttgctgcagt tcagcattag ccttggccgc tttggccctg gcctgcaaca
     5641 tatttttctt ggttttagct tcggaaattt ttgcttctag agccgcgagg ttgcgacgga
     5701 gattctccga catggtcctc tgttgggcta gctgggtttg gtacgccgcc gccgtatctg
     5761 tcagactttt tttgcgggcc agggcttccc tagccaaatt ttcttcccca ttggtgaggg
     5821 ctaattttgc ccggtcttcc catttcttgg cttcctgggt gtcttggtta agacgttgct
     5881 ctgttcgttt ttcttcggcg atcgtgcggg caacggcctg gcggagttgc accaggtctt
     5941 cctgcatatc gatcacggcc tgttccagaa ctttttctgg atcttc
//
