LOCUS       sequence-478376-        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(57..73)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(57..73)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(70..92)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(245..833)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(325..344)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(966..1060)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1081..1740)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1655..1674
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1741..1843)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     1930..1952
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1944..1961
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1945..1961
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(2050..2073,2074..2109,2110..2133)
                     /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"
     CDS             2140..2163
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
ORIGIN
        1 aggctaggtg gaggctcagt gatgataagt ctgcgatggt ggatgcatgt gtcatggtca
       61 tagctgtttc ctgtgtgaaa ttgttatccg ctcagagggc acaatcctat tccgcgctat
      121 ccgacaatct ccaagacatt aggtggagtt cagttcggcg tatggcatat gtcgctggaa
      181 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
      241 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
      301 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
      361 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
      421 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
      481 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
      541 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
      601 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
      661 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
      721 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
      781 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
      841 ctttgatctt ttctacgggg tctgacgctc tattcaacaa agccgccgtc ccgtcaagtc
      901 agcgtaaatg ggtagggggc ttcaaatcgt ccccccatac gatataagtt gttactagtg
      961 cttggattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa caaatccaga
     1021 tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct cgatatcaaa
     1081 ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac
     1141 atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc
     1201 gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc
     1261 cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt
     1321 ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga
     1381 atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt
     1441 ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc
     1501 accgtctttc attgccatac gaaattccgg atgagcattc atcaggcggg caagaatgtg
     1561 aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat
     1621 atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg
     1681 ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat
     1741 tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct
     1801 tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attgatacct
     1861 gaaacaaaac ccatcgtacg gccaaggaag tctccaataa ctgtgatcca ccacaagcgc
     1921 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tcatgcataa tccgcacgca
     1981 tctggaataa ggaagtgcca ttccgcctga cctgcatcgt ctcatcggtc tcaatccggt
     2041 ggtggttctt ggtcccatcc acaatttgaa aaaggtggtg gttctggtgg tggttctggt
     2101 ggttctgctt ggtcccatcc acaatttgaa aaaggatccg actataagga tgacgacgat
     2161 aaggctggat ctaagcgtga ggcggacgct gaggcatggc attggttaca attaaagcca
     2221 gggcagccaa tgtattaatg gctgagacct gagacggcat gtatgaccgc gtttcgagtt
     2281 gaagaaggaa acttatgatc tgccatacgg ggcac
//
