LOCUS       sequence-485233-        7335 bp    DNA     circular SYN 03-SEP-2025
DEFINITION  Bacterial expression of SynPspA with N-terminal NusA-tag and 3C 
            cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7335)
  AUTHORS   Junglas B, Huber ST, Heidler T, Schlosser L, Mann D, Hennig R, 
            Clarke M, Hellmann N, Schneider D, Sachse C
  TITLE     PspA adopts an ESCRT-III-like fold and remodels bacterial membranes.
  JOURNAL   Cell. 2021 Jul 8;184(14):3674-3688.e18. doi: 
            10.1016/j.cell.2021.05.042. Epub 2021 Jun 23.
  PUBMED    34166616
REFERENCE   2  (bases 1 to 7335)
  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: 
            "10.1016/j.cell.2021.05.042"; journalName: "Cell"; date: "2021-07-8-
            8"; volume: "184"; issue: "14"; pages: "3674-3688.e18"
COMMENT     Sequence Label: pET50b_SynPspA
FEATURES             Location/Qualifiers
     source          1..7335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             10..27
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             28..72
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(153..171)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      167..214
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      251..706
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(338..357)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     548..569
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(798..1613)
                     /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             complement(798..1613)
                     /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     1521..1540
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1605..1624
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1735..2323
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2224..2243
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2477..2494
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2509..2648
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2574..2593)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2734..2756
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2750..2941)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3513..4595)
                     /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
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4557..4576
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4596..4673)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4879..4898
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4986..5005
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4986..5004
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5005..5029
                     /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)."
     RBS             5044..5066
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5086..5103
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5110..6594
                     /codon_start=1
                     /gene="nusA"
                     /product="transcription elongation protein (N utilization 
                     substance protein A)"
                     /label=NusA
                     /note="highly soluble in E. coli"
                     /translation="MNKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDV
                     RVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDR
                     ITTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVIL
                     REDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIE
                     IKAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPA
                     QFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMT
                     VDDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLD
                     EPTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLED
                     LAEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA"
     CDS             6610..6627
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6637..6660
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 gagctcgctc tggtgccacg cggtagtaaa gaaaccgctg ctgctaaatt cgaacgccag
       61 cacatggaca gctctacttc tgctgctctc gaggcttaat taacctaggc tgctaaacaa
      121 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
      181 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
      241 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
      301 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
      361 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
      421 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
      481 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
      541 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
      601 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
      661 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
      721 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
      781 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
      841 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
      901 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
      961 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     1021 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     1081 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     1141 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcactgtt aaaaggacaa
     1201 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     1261 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttgccggg gatcgcagtg
     1321 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     1381 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     1441 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     1501 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     1561 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     1621 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     1681 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     1741 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     1801 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     1861 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     1921 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     1981 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2041 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2101 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2161 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2221 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2281 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2341 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     2401 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     2461 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     2521 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     2581 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     2641 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     2701 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     2761 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     2821 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     2881 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     2941 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     3001 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     3061 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     3121 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     3181 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     3241 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     3301 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     3361 agcctagccg ggtcctcaac gacaggagca cgatcatgct agtcatgccc cgcgcccacc
     3421 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     3481 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3541 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3601 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     3661 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     3721 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     3781 cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     3841 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     3901 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     3961 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     4021 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     4081 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     4141 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     4201 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     4261 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     4321 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     4381 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     4441 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     4501 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     4561 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     4621 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     4681 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     4741 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     4801 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     4861 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     4921 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc
     4981 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     5041 aattttgttt aactttaaga aggagatata catatgggca gcagccatca ccaccatcac
     5101 cattctagta tgaacaaaga aattttggct gtagttgaag ccgtatccaa tgaaaaggcg
     5161 ctacctcgcg agaagatttt cgaagcattg gaaagcgcgc tggcgacagc aacaaagaaa
     5221 aaatatgaac aagagatcga cgtccgcgta cagatcgatc gcaaaagcgg tgattttgac
     5281 actttccgtc gctggttagt tgttgatgaa gtcacccagc cgaccaagga aatcaccctt
     5341 gaagccgcac gttatgaaga tgaaagcctg aacctgggcg attacgttga agatcagatt
     5401 gagtctgtta cctttgaccg tatcactacc cagacggcaa aacaggttat cgtgcagaaa
     5461 gtgcgtgaag ccgaacgtgc gatggtggtt gatcagttcc gtgaacacga aggtgaaatc
     5521 atcaccggcg tggtgaaaaa agtaaaccgc gacaacatct ctctggatct gggcaacaac
     5581 gctgaagccg tgatcctgcg cgaagatatg ctgccgcgtg aaaacttccg ccctggcgac
     5641 cgcgttcgtg gcgtgctcta ttccgttcgc ccggaagcgc gtggcgcgca actgttcgtc
     5701 actcgttcca agccggaaat gctgatcgaa ctgttccgta ttgaagtgcc agaaatcggc
     5761 gaagaagtga ttgaaattaa agcagcggct cgcgatccgg gttctcgtgc gaaaatcgcg
     5821 gtgaaaacca acgataaacg tatcgatccg gtaggtgctt gcgtaggtat gcgtggcgcg
     5881 cgtgttcagg cggtgtctac tgaactgggt ggcgagcgta tcgatatcgt cctgtgggat
     5941 gataacccgg cgcagttcgt gattaacgca atggcaccgg cagacgttgc ttctatcgtg
     6001 gtggatgaag ataaacacac catggacatc gccgttgaag ccggtaatct ggcgcaggcg
     6061 attggccgta acggtcagaa cgtgcgtctg gcttcgcaac tgagcggttg ggaactcaac
     6121 gtgatgaccg ttgacgacct gcaagctaag catcaggcgg aagcgcacgc agcgatcgac
     6181 accttcacca aatatctcga catcgacgaa gacttcgcga ctgttctggt agaagaaggc
     6241 ttctcgacgc tggaagaatt ggcctatgtg ccgatgaaag agctgttgga aatcgaaggc
     6301 cttgatgagc cgaccgttga agcactgcgc gagcgtgcta aaaatgcact ggccaccatt
     6361 gcacaggccc aggaagaaag cctcggtgat aacaaaccgg ctgacgatct gctgaacctt
     6421 gaaggggtag atcgtgattt ggcattcaaa ctggccgccc gtggcgtttg tacgctggaa
     6481 gatctcgccg aacagggcat tgatgatctg gctgatatcg aagggttgac cgacgaaaaa
     6541 gccggagcac tgattatggc tgcccgtaat atttgctggt tcggtgacga agcgactagt
     6601 ggttctggtc atcaccatca ccatcactcc gcggctcttg aagtcctctt tcagggaccc
     6661 gggatggaac tatttaatag ggttggaaga gtattgaagt cccaattgac ccattggcag
     6721 cagcagcaag aagctccgga agatctgttg gagcgcctgc tgggcgaaat ggaattggag
     6781 ctgatcgagc tgcgtcgcgc actggcgcag accattgcga cgtttaaaag cactgagcgc
     6841 caacgtgatg cgcaacagct gattgcccaa cgttggtacg aaaaggcgca ggcggcgttg
     6901 gaccgtggta atgagcaact agctcgtgaa gcgctgggtc agcgtcagag ctatcagtcc
     6961 cacaccgaag cgctcggcaa atctctgggt gaacaacgtg cattggtcga gcaggttaga
     7021 ggccagctgc aaaagctgga gcgtaaatac ctggagctta agagccagaa gaacctttat
     7081 ctggcccgcc tgaagtcagc aattgctgcg cagaagatcg aggagatcgc gggtaacctg
     7141 gacaacgcga gcgcaagctc tctgttcgaa cggattgaaa ccaaaatcct ggagttagag
     7201 gccgaacgtg aactgttgaa tccgcctccg tccccgctgg acaaaaaatt cgagcagtgg
     7261 gaagagcaac aagctgtgga agcaaccctg gctgccatga aagcgcgccg tagcttaccg
     7321 ccgccaagct cgtaa
//
