LOCUS       sequence-448136-        5875 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SynPspA R44K/E126Q/E179Q 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 5875)
  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 5875)
  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/s41594-024-01359-7"; journalName: "Nat Struct Mol Biol"; 
            date: "2025-01"; volume: "32"; issue: "1"; pages: "23-34"
COMMENT     Sequence Label: pET50del_SynPspA_R44K/E126Q/E179Q
FEATURES             Location/Qualifiers
     source          1..5875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(111..130)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             122..937
                     /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             122..937
                     /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(195..214)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(1029..1484)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1166..1187)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1378..1397
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1521..1568)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1564..1582
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1663..1707)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(1708..1725)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2468..2491)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(2507..2524)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2544..2566
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2581..2605
                     /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(2605..2624)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2606..2624)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2712..2731)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2937..3014
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3015..4097
                     /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(3034..3053)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4669..4860
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4854..4876)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4962..5101
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5017..5036
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5116..5133)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5287..5875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5367..5386)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
      121 tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga
      181 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
      241 ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
      301 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
      361 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
      421 gatccccggc aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
      481 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
      541 ttttaacagt gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
      601 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
      661 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
      721 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
      781 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
      841 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
      901 attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca
      961 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     1021 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     1081 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     1141 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     1201 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     1261 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     1321 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     1381 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     1441 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg
     1501 gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt
     1561 tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg ggctttgttt
     1621 agcagcctag gttaattaag cctcgagagc agcagaagta gagctgtcca tgtgctggcg
     1681 ttcgaattta gcagcagcgg tttctttact accgcgtggc accagagcga gctctgcggc
     1741 cgcaagcttg tcgacggacg tcgggcgcgc caaggcctgt acagaattcg gatccttacg
     1801 agcttggcgg cggtaagcta cggcgcgctt tcatggcagc cagggttgct tccacagctt
     1861 gttgctcttc ccactgctcg aattttttgt ccagcgggga cggaggcgga ttcaacagtt
     1921 cacgttcggc ctgtaactcc aggattttgg tttcaatccg ttcgaacaga gagcttgcgc
     1981 tcgcgttgtc caggttaccc gcgatctcct cgatcttctg cgcagcaatt gctgacttca
     2041 ggcgggccag ataaaggttc ttctggctct taagctccag gtatttacgc tgcagctttt
     2101 gcagctggcc tctaacctgc tcgaccaatg cacgttgttc acccagagat ttgccgagcg
     2161 cttcggtgtg ggactgatag ctctgacgct gacccagcgc ttcacgagct agttgctcat
     2221 taccacggtc caacgccgcc tgcgcctttt cgtaccaacg ttgggcaatc agctgttgcg
     2281 catcacgttg gcgctcagtg cttttaaacg tcgcaatggt ctgcgccagt gcgcgtttca
     2341 gctcgatcag ctccaattcc atttcgccca gcaggcgctc caacagatct tccggagctt
     2401 cttgctgctg ctgccaatgg gtcaattggg acttcaatac tcttccaacc ctattaaata
     2461 gttccatggg tccctgaaag aggacttcaa gagccgcgga actagaatgg tgatggtggt
     2521 gatggctgct gcccatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg
     2581 ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag
     2641 atcgatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
     2701 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
     2761 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
     2821 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
     2881 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca
     2941 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     3001 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     3061 cttatcagac cgtttcccgc gtggtaaacc aggccagcca cgtttctgcg aaaacgcggg
     3121 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     3181 tggcgggcaa acagtcgttg ctgattggcg ttaccacctc cagtctggcc ctgcacgcgc
     3241 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     3301 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     3361 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     3421 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     3481 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     3541 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     3601 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     3661 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     3721 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     3781 ccattaccga gtccgggctg cgcgttggtg cggacatctc ggtagtggga tacgacgata
     3841 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     3901 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     3961 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     4021 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     4081 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg
     4141 ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
     4201 ggcatgacta gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc
     4261 cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc
     4321 aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt
     4381 ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc
     4441 tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga
     4501 gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc
     4561 agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc
     4621 ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag
     4681 gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag
     4741 aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac
     4801 gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     4861 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     4921 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     4981 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga
     5041 gagtgcacca ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     5101 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5161 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5221 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5281 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5341 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5401 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5461 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5521 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5581 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5641 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5701 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     5761 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5821 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaa
//
