LOCUS       sequence-448196-        5875 bp    DNA     circular SYN 15-APR-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 5875)
  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 5875)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 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..5875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     831..850
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(870..947)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1153..1172
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1260..1279
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1260..1278
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1279..1303
                     /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             1318..1340
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1360..1377
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1393..1416
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2159..2176
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2177..2221
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2302..2320)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2316..2363
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2400..2855
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2487..2506)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2697..2718
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2947..3762)
                     /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(2947..3762)
                     /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     3670..3689
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3754..3773
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3884..4472
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4373..4392
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4626..4643
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4658..4797
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4723..4742)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4883..4905
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4899..5090)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5662..5875,1..869))
                     /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"
ORIGIN
        1 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
       61 accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
      121 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
      181 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
      241 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
      301 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
      361 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
      421 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
      481 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
      541 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
      601 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
      661 ctggaggtgg taacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
      721 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
      781 acgtggctgg cctggtttac cacgcgggaa acggtctgat aagagacacc ggcatactct
      841 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
      901 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
      961 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     1021 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     1081 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     1141 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     1201 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc ccgcgaaatt
     1261 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
     1321 gtttaacttt aagaaggaga tatacatatg ggcagcagcc atcaccacca tcaccattct
     1381 agttccgcgg ctcttgaagt cctctttcag ggacccatgg aactatttaa tagggttgga
     1441 agagtattga agtcccaatt gacccattgg cagcagcagc aagaagctcc ggaagatctg
     1501 ttggagcgcc tgctgggcga aatggaattg gagctgatcg agctgcgtcg cgcactggcg
     1561 cagaccattg cgacgtttaa aagcactgag cgccaacgtg atgcgcaaca gctgattgcc
     1621 caacgttggt acgaaaaggc gcaggcggcg ttggaccgtg gtaatgagca actagctcgt
     1681 gaagcgctgg gtcagcgtca gagctatcag tcccacaccg aagcgctcgg caaatctctg
     1741 ggtgaacaac gtgcattggt cgagcaggtt agaggccagc tgcaaaagct ggagcgtaaa
     1801 tacctggagc ttaagagcca gaagaacctt tatctggccc gcctgaagtc agcaattgct
     1861 gcgcagaaga tcgaggagat cgcgggtaac ctggacaacg cgagcgcaag ctctctgttc
     1921 gaacggattg aaaccaaaat cctggagtta gaggccgaac gtgaactgtt gaatccgcct
     1981 ccgtccccgc tggacaaaaa attcgagcag tgggaagagc aacaagctgt ggaagcaacc
     2041 ctggctgcca tgaaagcgcg ccgtagctta ccgccgccaa gctcgtaagg atccgaattc
     2101 tgtacaggcc ttggcgcgcc cgacgtccgt cgacaagctt gcggccgcag agctcgctct
     2161 ggtgccacgc ggtagtaaag aaaccgctgc tgctaaattc gaacgccagc acatggacag
     2221 ctctacttct gctgctctcg aggcttaatt aacctaggct gctaaacaaa gcccgaaagg
     2281 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     2341 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     2401 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2461 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2521 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2581 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2641 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2701 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2761 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2821 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     2881 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     2941 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     3001 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     3061 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     3121 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     3181 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     3241 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     3301 tgattgcgcc tgagcgagac gaaatacgcg atcactgtta aaaggacaat tacaaacagg
     3361 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     3421 aggatattct tctaatacct ggaatgctgt tttgccgggg atcgcagtgg tgagtaacca
     3481 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     3541 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     3601 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     3661 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     3721 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     3781 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     3841 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3901 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3961 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4021 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4081 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4141 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4201 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4261 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4321 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4381 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4441 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4501 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4561 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4621 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4681 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     4741 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     4801 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4861 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4921 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     4981 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     5041 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     5101 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     5161 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     5221 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     5281 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     5341 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     5401 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     5461 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     5521 gtcctcaacg acaggagcac gatcatgcta gtcatgcccc gcgcccaccg gaaggagctg
     5581 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     5641 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5701 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     5761 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     5821 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgt
//
