LOCUS       sequence-197242-        3809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a fusion protein of KcsA-Kv1.3 and sfGFP with a C-terminal
            His tag. The expression is controlled by a OR2-OR1-Pr promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3809)
  AUTHORS   Guo S, Vaish A, Chen Q, Murray RM
  TITLE     Expressing Biologically Active Membrane Proteins in a Cell-Free 
            Transcription-Translation Platform
  JOURNAL   bioRxiv 104455
REFERENCE   2  (bases 1 to 3809)
  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: "bioRxiv 
            104455"
COMMENT     Sequence Label: pSG66-KcsA-Kv1.3-sfGFP
FEATURES             Location/Qualifiers
     source          1..3809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             7..29
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             534..554
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             585..1298
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             1299..1316
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     rep_origin      complement(1668..2256)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1748..1767)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2427..3218,3219..3287))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLHVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRHEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHATRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3050..3069
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3288..3392)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3460..3478
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aataattttg tttaacttta agaaggagat atacaatgga cgaagttgat ggtccgccaa
       61 tgttgtccgg tctgcttgct cgtctggtta agttgcttct gggccgtcac ggttctgcgc
      121 tgcactggcg tgctgcaggc gctgccaccg tccttctggt gatcgtactg cttgctggtt
      181 cctacttggc ggttctggcg gaagcggatg atccgaccag cggctttagc agcattccgg
      241 acgccttgtg gtggtctgtc gataccgcta ctaccgtggg ttacggcgac ttgtatccgg
      301 tgaccctttg gggtcgtctg gtagcggttg tcgttatggt ggctggcatc acctccttcg
      361 gtctggtaac tgctgccctt gctacttggt ttgtaggccg cgaacaagag cgtcgtggtc
      421 atttcgttcg tcactctgaa aaagcggctg aggaggcgta cacccgtact acccgtgctc
      481 tgcacgagcg ctttgaccgt ctggaacgta tgcttgacga taaccgtcgt ggtgaaaacc
      541 tgtacttcca gtctggtgca ttgtacttcc agtctggtgg tgctatgcgt aaaggagaag
      601 aacttttcac tggtgtcgtc cctattctgg tggaactgga tggtgatgtc aacggtcata
      661 agttttccgt gcgtggcgag ggtgaaggtg acgcaactaa tggtaaactg acgctgaagt
      721 tcatctgtac tactggtaaa ctgccggtac cttggccgac tctggtaacg acgctgactt
      781 atggtgttca gtgctttgct cgttatccgg accatatgaa gcagcatgac ttcttcaagt
      841 ccgccatgcc ggaaggctat gtgcaggaac gcacgatttc ctttaaggat gacggcacgt
      901 acaaaacgcg tgcggaagtg aaatttgaag gcgataccct ggtaaaccgc attgagctga
      961 aaggcattga ctttaaagaa gatggcaata tcctgggcca taagctggaa tacaatttta
     1021 acagccacaa tgtttacatc accgccgata aacaaaaaaa tggcattaaa gcgaatttta
     1081 aaattcgcca caacgtggag gatggcagcg tgcagctggc tgatcactac cagcaaaaca
     1141 ctccaatcgg tgatggtcct gttctgctgc cagacaatca ctatctgagc acgcaaagcg
     1201 ttctgtctaa agatccgaac gagaaacgcg atcatatggt tctgctggag ttcgtaaccg
     1261 cagcgggcat cacgcatggt atggatgaac tgtacaaaca tcatcatcat catcattgaa
     1321 ctcgagcaaa gcccgccgaa aggcgggctt ttctgtgtcg accgatgccc ttgagagcct
     1381 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     1441 ctgtgttctt tatcatgcaa ctcgtaggac aggtgccggc agctctcttc cgcttcctcg
     1501 ctcacttact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     1561 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     1621 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     1681 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     1741 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     1801 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1861 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1921 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1981 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2041 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2101 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2161 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2221 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2281 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     2341 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2401 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2461 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2521 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgtga cccacgctca
     2581 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     2641 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     2701 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     2761 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttgca
     2821 tgatccccca tgttgtgcaa aaaagctgtt agctccttcg gtcctccgat cgttgtcaga
     2881 agtaagttgg ccgcagtgtt atcactcatt gttatggcag cactgcataa ttctcttact
     2941 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3001 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg
     3061 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcgtg gcgaaaactc
     3121 tcaaggatct taccgctgtt tagatccagt tcgatgtaac ccactcgtgc acccaactga
     3181 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacatg aaggcaaaat
     3241 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3301 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3361 atttagaaaa ataaacaaat aggtgttccg cgcacatttc cccgaaaagt gccacctgac
     3421 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     3481 tttcgtgttc aagaattctg gcgaatcctc ttaccagcca gaaaacgacc tttctgtggt
     3541 gaaaccggat gctgcaattc agagcgccag caagtggggg acagcagatg acctgaccgc
     3601 cgcagagtgg atgtttgaca tggtgatgac tatcgcacca tcagccagaa aaccgaattt
     3661 tgctgggtgg gctaacgata tccgcctgat gcgtgaacgt gacggacgta accaccgcga
     3721 catgtgtgtg ctgttccgct gggcatgctg agctaacacc gtgcgtgttg acaattttac
     3781 ctctggcggt gataatggtt gcagctagc
//
