LOCUS       sequence-425807-        3176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cm NCPPB382 cspA2 codon-optimized for E. coli recombinant protein 
            expression (6xHis-CspA2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3176)
  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 3176)
  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-CspA2
FEATURES             Location/Qualifiers
     source          1..3176
                     /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(1247..1271)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1360..1378)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1374..1421
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1518..1973
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1605..1624)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1815..1836
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1999..2103
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2104..2172,2173..2964)
                     /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(2322..2341)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3135..3176,1..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 ggcttcatgg caaatggaac tgttaaatgg tttaatggcg agaaagggtt tggtttcata
     1081 accgtggatg cagtagaagg aggccccgcg cagcaagacg ttttcgttca ttattctgcc
     1141 atcgaaatgt ctgggtataa agtgttggaa gagggccaac gtgtggcatt tgagattggt
     1201 caaggttcaa aaggtttaca ggccgagaat gtgacattgg cgtaggaccc agctttcttg
     1261 tacaaagtgg tgctcgagat ctgcagctgg taccatggaa ttcgaagctt gatccggctg
     1321 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     1381 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1441 ccggatctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     1501 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1561 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1621 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     1681 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     1741 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     1801 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     1861 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     1921 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     1981 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2041 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2101 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2161 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2221 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2281 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     2341 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     2401 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     2461 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     2521 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     2581 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     2641 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     2701 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     2761 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     2821 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     2881 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     2941 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3001 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3061 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3121 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaac
//
