LOCUS       sequence-425806-        3161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cm NCPPB382 cspA1 codon-optimized for E. coli recombinant protein 
            expression (6xHis-CspA1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3161)
  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 3161)
  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-CspA1
FEATURES             Location/Qualifiers
     source          1..3161
                     /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(1232..1256)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1345..1363)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1359..1406
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1503..1958
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1590..1609)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1800..1821
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1984..2088
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2089..2157,2158..2949)
                     /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(2307..2326)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3120..3161,1..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 cgacaggtac tgtaaaatgg tttaatgcgg aaaagggatt cgggttcatc
     1081 gcgccagaca atggaacagc tgatgttttc gctcattact ctgccatcgc cacaggtggt
     1141 tacaaatctt tggacgaaaa ccaaaaggtg gagtttgaag tagcacaagg tcctaaaggt
     1201 ccgcaagctg agaatatccg ccctttgtaa gacccagctt tcttgtacaa agtggtgctc
     1261 gagatctgca gctggtacca tggaattcga agcttgatcc ggctgctaac aaagcccgaa
     1321 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1381 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga tctggcgtaa
     1441 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     1501 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1561 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1621 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1681 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1741 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1801 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     1861 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     1921 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     1981 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2041 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2101 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2161 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2221 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     2281 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     2341 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     2401 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     2461 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     2521 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     2581 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     2641 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     2701 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     2761 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     2821 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     2881 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     2941 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3001 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3061 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3121 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa c
//
