LOCUS       sequence-448072-        5875 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of SynPspA 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
FEATURES             Location/Qualifiers
     source          1..5875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(673..696)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(712..729)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             749..771
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    786..810
                     /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(810..829)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(811..829)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(917..936)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1142..1219
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1220..2302
                     /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(1239..1258)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2874..3065
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3059..3081)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3167..3306
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3222..3241
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3321..3338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3492..4080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3572..3591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4191..4210)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4202..5017
                     /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             4202..5017
                     /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(4275..4294)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5109..5564)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5246..5267)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5458..5477
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5601..5648)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5644..5662
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5743..5787)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(5788..5805)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 ttacgagctt ggcggcggta agctacggcg cgctttcatg gcagccaggg ttgcttccac
       61 agcttgttgc tcttcccact gctcgaattt tttgtccagc ggggacggag gcggattcaa
      121 cagttcacgt tcggcctcta actccaggat tttggtttca atccgttcga acagagagct
      181 tgcgctcgcg ttgtccaggt tacccgcgat ctcctcgatc ttctgcgcag caattgctga
      241 cttcaggcgg gccagataaa ggttcttctg gctcttaagc tccaggtatt tacgctccag
      301 cttttgcagc tggcctctaa cctgctcgac caatgcacgt tgttcaccca gagatttgcc
      361 gagcgcttcg gtgtgggact gatagctctg acgctgaccc agcgcttcac gagctagttg
      421 ctcattacca cggtccaacg ccgcctgcgc cttttcgtac caacgttggg caatcagctg
      481 ttgcgcatca cgttggcgct cagtgctttt aaacgtcgca atggtctgcg ccagtgcgcg
      541 acgcagctcg atcagctcca attccatttc gcccagcagg cgctccaaca gatcttccgg
      601 agcttcttgc tgctgctgcc aatgggtcaa ttgggacttc aatactcttc caaccctatt
      661 aaatagttcc atgggtccct gaaagaggac ttcaagagcc gcggaactag aatggtgatg
      721 gtggtgatgg ctgctgccca tatgtatatc tccttcttaa agttaaacaa aattatttct
      781 agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga
      841 tcgagatcga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
      901 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
      961 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
     1021 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc
     1081 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc
     1141 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     1201 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     1261 tgtctcttat cagaccgttt cccgcgtggt aaaccaggcc agccacgttt ctgcgaaaac
     1321 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     1381 acaactggcg ggcaaacagt cgttgctgat tggcgttacc acctccagtc tggccctgca
     1441 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     1501 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     1561 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     1621 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     1681 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     1741 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     1801 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     1861 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     1921 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     1981 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggac atctcggtag tgggatacga
     2041 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     2101 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     2161 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     2221 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     2281 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     2341 caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     2401 cgcggggcat gactagcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg
     2461 gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct
     2521 gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt
     2581 aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag
     2641 gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac
     2701 cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac
     2761 gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt
     2821 tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca
     2881 aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc
     2941 tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg
     3001 accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
     3061 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     3121 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
     3181 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt
     3241 actgagagtg caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     3301 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3361 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3421 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3481 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3541 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3601 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3661 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3721 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3781 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3841 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3901 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     3961 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4021 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4081 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4141 gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     4201 tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga
     4261 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     4321 ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     4381 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     4441 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     4501 gatccccggc aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     4561 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     4621 ttttaacagt gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     4681 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     4741 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     4801 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     4861 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     4921 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     4981 attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca
     5041 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5101 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     5161 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     5221 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     5281 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     5341 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     5401 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     5461 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     5521 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg
     5581 gatatagttc ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt
     5641 tatgctagtt attgctcagc ggtggcagca gccaactcag cttcctttcg ggctttgttt
     5701 agcagcctag gttaattaag cctcgagagc agcagaagta gagctgtcca tgtgctggcg
     5761 ttcgaattta gcagcagcgg tttctttact accgcgtggc accagagcga gctctgcggc
     5821 cgcaagcttg tcgacggacg tcgggcgcgc caaggcctgt acagaattcg gatcc
//
