LOCUS       sequence-425808-        3340 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cm NCPPB382 cspB codon-optimized for E. coli recombinant protein 
            expression (6xHis-CspB).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3340)
  AUTHORS   Stevens DM, Moreno-Perez A, Weisberg AJ, Ramsing C, Fliegmann J, 
            Zhang N, Madrigal M, Martin G, Steinbrenner A, Felix G, Coaker G
  TITLE     Natural variation of immune epitopes reveals intrabacterial 
            antagonism.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Jun 4;121(23):e2319499121. doi: 
            10.1073/pnas.2319499121. Epub 2024 May 30.
  PUBMED    38814867
REFERENCE   2  (bases 1 to 3340)
  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"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2024-06-4"; volume: "121(23):e2319499121. 
            doi"; pages: " 10.1073/pnas.2319499121. Epub 2024 May 30"
COMMENT     Sequence Label: pRSET-Cm-CspB
FEATURES             Location/Qualifiers
     source          1..3340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     803..822
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        803..821
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             853..875
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             895..912
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             916..948
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(952..960,961..975)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     protein_bind    1000..1024
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1411..1435)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1524..1542)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1538..1585
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1682..2137
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1769..1788)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1979..2000
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2163..2267
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2268..2336,2337..3128)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2486..2505)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3299..3340,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      781 caggatctcg atcccgcgaa attaatacga ctcactatag ggagaccaca acggtttccc
      841 tctagaaata attttgttta actttaagaa ggagatatac atatgcgggg ttctcatcat
      901 catcatcatc atggtatggc tagcatgact ggtggacagc aaatgggtcg ggatctgtac
      961 gacgatgacg ataaggatcg atggggatcc gagctcgaga caagtttgta caaaaaagca
     1021 ggcttcatgc caactgggaa agtcaaattt tatgatgaag acaaggggtt tgggtttata
     1081 tcgtccgacg atggacagga agtcttcctg cacgcctccg cattacccag tggagtcgca
     1141 ggcgtgaaag ctggtactcg cttagagttc ggcatagccg acggaaaaag aggggcacag
     1201 gctttgagcg cccgcatact tgacgccccc ccgagtctgg ctcgcatgtc cagaaaaccc
     1261 gcagacgata tggctgtaat tgttgaagat ttggtaaagg tgttagacgg cataggcaca
     1321 ggtttaaaga gaggcagata ccctgaagat tcccacggac gtaaaatagc tgcacttctt
     1381 cggcgggtcg ccgaggaact ggacgcgtga acccagcttt cttgtacaaa gtggtgctcg
     1441 agatctgcag ctggtaccat ggaattcgaa gcttgatccg gctgctaaca aagcccgaaa
     1501 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1561 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat ctggcgtaat
     1621 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     1681 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1741 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1801 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1861 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     1921 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     1981 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2041 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2101 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     2161 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2221 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     2281 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     2341 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     2401 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     2461 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     2521 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     2581 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     2641 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     2701 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     2761 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     2821 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     2881 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     2941 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     3001 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     3061 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     3121 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     3181 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     3241 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3301 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
//
