LOCUS       sequence-482778-        2315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid insert contains the aMFx1, FLAG and twinStrep C-terminal tag
            sequences for use in Combinatorial Golden Gate Assembly (BsaI 
            restriction sites)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2315)
  AUTHORS   Cleaver A, Luo R, Smith OB, Murphy L, Schwessinger B, Brock J
  TITLE     High-throughput optimisation of protein secretion in yeast via an 
            engineered biosensor.
  JOURNAL   Trends Biotechnol. 2025 Apr;43(4):838-867. doi: 
            10.1016/j.tibtech.2024.11.010. Epub 2024 Dec 13.
  PUBMED    39674781
REFERENCE   2  (bases 1 to 2315)
  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"; doi: 
            "10.1016/j.tibtech.2024.11.010"; journalName: "Trends Biotechnol"; 
            date: "2025-04"; volume: "43"; issue: "4"; pages: "838-867"
COMMENT     Sequence Label: twinStrep_FLAG_aMFx1 (4a)
FEATURES             Location/Qualifiers
     source          1..2315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(5..22)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5..21)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14..36)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        123..225
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             226..885
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(292..311)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      906..1000
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1133..1721
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1622..1641
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1874..1896
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1893..1909
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1893..1909
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(2118..2141)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(join(2148..2171,2172..2207,2208..2231))
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
ORIGIN
        1 catgactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgct tgtggtggat
       61 cacagttatt ggagacttcc ttggccgtac gatgggtttt gtttcaggta tcaatgagac
      121 gttgatcggc acgtaagagg ttccaacttt caccataatg aaataagatc actaccgggc
      181 gtattttttg agttatcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc
      241 actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt
      301 cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta
      361 aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc
      421 ctgatgaatg ctcatccgga atttcgtatg gcaatgaaag acggtgagct ggtgatatgg
      481 gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc
      541 tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg
      601 tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc
      661 tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac
      721 ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg
      781 ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt
      841 aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaatttga tatcgagctc
      901 gcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag
      961 aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaagcacta gtaacaactt
     1021 atatcgtatg gggggacgat ttgaagcccc ctacccattt acgctgactt gacgggacgg
     1081 cggctttgtt gaatagagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     1141 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     1201 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     1261 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     1321 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     1381 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     1441 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     1501 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     1561 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     1621 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     1681 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     1741 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcca gcgacatatg
     1801 ccatacgccg aactgaactc cacctaatgt cttggagatt gtcggatagc gcggaatagg
     1861 attgtgccct ctgagcggat aacaatttca cacaggaaac agctatgacc atgacacatg
     1921 catccaccat cgcagactta tcatcactga gcctccacct agcctgtgcc ccgtatggca
     1981 gatcataagt ttccttcttc aactcgaaac gcggtcatac atgccgtctc aggtctcagc
     2041 cattaataca ttggctgccc tggctttaat tgtaaccaat gccatgcctc agcgtccgcc
     2101 tcacgcttag atccagcctt atcgtcgtca tccttatagt cggatccttt ttcaaattgt
     2161 ggatgggacc aagcagaacc accagaacca ccaccagaac caccaccttt ttcaaattgt
     2221 ggatgggacc aagaaccacc accggattga gaccgatgag acgatgcagg tcaggcggaa
     2281 tggcacttcc ttattccaga tgcgtgcgga ttatg
//
