LOCUS       sequence-485300-        5806 bp    DNA     circular SYN 04-SEP-2025
DEFINITION  Bacterial expression of SynPspA H1-5 with N-terminal 6xHis-Tag and 
            3C cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5806)
  AUTHORS   Junglas B, Hudina E, Schonnenbeck P, Ritter I, Heddier A, 
            Santiago-Schubel B, Huesgen PF, Schneider D, Sachse C
  TITLE     Structural plasticity of bacterial ESCRT-III protein PspA in 
            higher-order assemblies.
  JOURNAL   Nat Struct Mol Biol. 2025 Jan;32(1):23-34. doi: 
            10.1038/s41594-024-01359-7. Epub 2024 Aug 16.
  PUBMED    39152237
REFERENCE   2  (bases 1 to 5806)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41594-024-01359-7"; journalName: "Nat Struct Mol Biol"; 
            date: "2025-01"; volume: "32"; issue: "1"; pages: "23-34"
COMMENT     Sequence Label: pET50del_SynPspA_H1-5
FEATURES             Location/Qualifiers
     source          1..5806
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    35..174
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     90..109
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(189..206)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(360..948)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(440..459)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1059..1078)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1070..1885
                     /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             1070..1885
                     /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(1143..1162)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(1977..2432)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2114..2135)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2326..2345
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2469..2516)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2512..2530
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2611..2655)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(2656..2673)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(3347..3370)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(3386..3403)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3423..3445
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3460..3484
                     /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(3484..3503)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3485..3503)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3591..3610)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3816..3893
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3894..4976
                     /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
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSV
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3913..3932)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5548..5739
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5733..5755)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
       61 atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
      121 ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
      181 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
      241 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
      301 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
      361 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
      421 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
      481 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
      541 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
      601 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
      661 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
      721 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
      781 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
      841 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
      901 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
      961 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     1021 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
     1081 tcaacgggaa acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata
     1141 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta
     1201 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     1261 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     1321 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccggcaa
     1381 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     1441 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagtga
     1501 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     1561 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     1621 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     1681 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     1741 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     1801 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     1861 tttgatgctc gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta
     1921 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     1981 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     2041 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     2101 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     2161 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     2221 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     2281 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     2341 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     2401 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccaatccgga tatagttcct
     2461 cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat
     2521 tgctcagcgg tggcagcagc caactcagct tcctttcggg ctttgtttag cagcctaggt
     2581 taattaagcc tcgagagcag cagaagtaga gctgtccatg tgctggcgtt cgaatttagc
     2641 agcagcggtt tctttactac cgcgtggcac cagagcgagc tctgcggccg caagcttgtc
     2701 gacggacgtc gggcgcgcca aggcctgtac agaattcgga tccttacgag cttggcggcg
     2761 gtaagctacg gcgcgctttc atggcagcca gggttgcttc cacagcttgt tgctcttccc
     2821 actgctcgaa ttttttgtcc agcggggacg gaggcggatt caacagttca cgttcggcct
     2881 ctaactccag gattttggtt tcaatccgtt cgaacagaga gcttgcgctc gcgttgtcca
     2941 ggttacccgc gatctcctcg atcttctgcg cagcaattgc tgacttcagg cgggccagat
     3001 aaaggttctt ctggctctta agctccaggt atttacgctc cagcttttgc agctggcctc
     3061 taacctgctc gaccaatgca cgttgttcac ccagagattt gccgagcgct tcggtgtggg
     3121 actgatagct ctgacgctga cccagcgctt cacgagctag ttgctcatta ccacggtcca
     3181 acgccgcctg cgccttttcg taccaacgtt gggcaatcag ctgttgcgca tcacgttggc
     3241 gctcagtgct tttaaacgtc gcaatggtct gcgccagtgc gcgacgcagc tcgatcagct
     3301 ccaattccat ttcgcccagc aggcgctcca acagatcttc cggagcgggt ccctgaaaga
     3361 ggacttcaag agccgcggaa ctagaatggt gatggtggtg atggctgctg cccatatgta
     3421 tatctccttc ttaaagttaa acaaaattat ttctagaggg gaattgttat ccgctcacaa
     3481 ttcccctata gtgagtcgta ttaatttcgc gggatcgaga tcgatctcga tcctctacgc
     3541 cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc
     3601 cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg
     3661 cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc
     3721 accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat
     3781 gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct
     3841 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
     3901 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
     3961 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     4021 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
     4081 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
     4141 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     4201 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     4261 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     4321 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     4381 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     4441 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     4501 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgtca
     4561 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
     4621 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
     4681 gcgttggtgc ggacatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
     4741 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     4801 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     4861 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
     4921 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     4981 aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg
     5041 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactag catgatcgtg
     5101 ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa
     5161 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca
     5221 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg
     5281 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
     5341 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc
     5401 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca
     5461 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac
     5521 agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg
     5581 gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg
     5641 gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc
     5701 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     5761 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtc
//
