LOCUS       sequence-323020-        3698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of sfGFP under control of PalkB promoter. Sensor output 
            plasmid for detection of n-butanol and branched-chain higher 
            alcohols..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3698)
  AUTHORS   Bahls MO, Platz L, Morgado G, Schmidt GW, Panke S
  TITLE     Directed evolution of biofuel-responsive biosensors for automated 
            optimization of branched-chain alcohol biosynthesis.
  JOURNAL   Metab Eng. 2021 Nov 9. pii: S1096-7176(21)00171-3. doi: 
            10.1016/j.ymben.2021.10.014.
  PUBMED    34767976
REFERENCE   2  (bases 1 to 3698)
  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: "Metab Eng. 
            2021 Nov 9. pii: S1096-7176(21)00171-3. doi: 
            10.1016/j.ymben.2021.10.014."
COMMENT     Sequence Label: pAB_PalkB_sfgfp_KanR
FEATURES             Location/Qualifiers
     source          1..3698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(132..134,135..845)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             132..845
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(162..190)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     785..805
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      869..955
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             1135..1326
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1320..1342)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1428..1568
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1483..1502
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1583..1600)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1754..2342)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1834..1853)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     oriT            2378..2486
                     /note="incP origin of transfer"
     CDS             complement(2653..3468)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3460..3479
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      complement(3600..3694)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 actagtctgg tttttccagc agacgacgga gcaaaaacta cccgtaggtg tagttggcgc
       61 aagcgtccga ttagctcagg ttttaagatg gaattcacac tttacttttt aattagcagg
      121 agatttaaca tatgagcaaa ggagaagaac ttttcactgg agttgtccca attcttgttg
      181 aattagatgg tgatgttaat gggcacaaat tttctgtcag aggagagggt gaaggtgatg
      241 ctacaaacgg aaaactcacc cttaaattta tttgcactac tggaaaacta cctgttccat
      301 ggccaacact tgtcactact ctgacctatg gtgttcaatg cttttcccgt tatccggatc
      361 acatgaaacg gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaacgca
      421 ctatatcttt caaagatgac gggacctaca agacgcgtgc tgaagtcaag tttgaaggtg
      481 atacccttgt taatcgtatc gagttaaaag gtattgattt taaagaagat ggaaacattc
      541 tcggacacaa actcgagtac aactttaact cacacaatgt atacatcacg gcagacaaac
      601 aaaagaatgg aatcaaagct aacttcaaaa ttcgccacaa cgttgaagat ggttccgttc
      661 aactagcaga ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag
      721 acaaccatta cctgtcgaca caatctgtcc tttcgaaaga tcccaacgaa aagcgtgacc
      781 acatggtcct tcttgagttt gtaactgctg ctgggattac acatggcatg gatgagctct
      841 acaaaggatc ctaattaatt aaaggcatca aataaaacga aaggctcagt cgaaagactg
      901 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
      961 gccctagaca gctgggcgcg cccccgccgc atccataccg ccagttgttt accctcacaa
     1021 cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt
     1081 ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc
     1141 aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt
     1201 ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgctgcac
     1261 gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     1321 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     1381 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
     1441 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg
     1501 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     1561 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     1621 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     1681 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     1741 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     1801 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     1861 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     1921 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     1981 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2041 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2101 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2161 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     2221 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2281 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2341 aagaagatcc tttgatcttt tctacggggc cggcctacgg ccagcctcgc agagcaggat
     2401 tcccgttgag caccgccagg tgcgaataag ggacagtgaa gaaggaacac ccgctcgcgg
     2461 gtgggcctac ttcacctatc ctgcccggct gacgccgttg gatacaccaa ggaaagtcta
     2521 cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat accgaaaaaa
     2581 tcgctataat gaccccgaag cagggttatg cagcggaaaa ggacaacgcg cggaccgcgg
     2641 tccaattaat tattagaaaa attcatccag catcagatga aattgcagtt tgttcatatc
     2701 cggattatca atgccatatt tctgaaacag acgtttttgc aggctcgggc taaattcgcc
     2761 caggcagttc cacagaatgg ccagatcctg ataacgatcc gcaatgccca cacggcccac
     2821 atcaatgcag ccaatcagtt tgccttcatc gaaaatcagg ttatccaggc taaaatcgcc
     2881 gtgggtcacc acgctatccg ggctaaacgg cagcagttta tgcatttctt tccacacctg
     2941 ttccaccggc cagccgttac gttcatcatc aaaatcgctc gcatccacca ggccgttgtt
     3001 catacggctc tgcgcctggg ccagacgaaa cacacgatcg ctgttaaacg ggcagttgca
     3061 caccggaatg ctatgcagac gacgcagaaa cacggccagc gcatccacaa tgttttcgcc
     3121 gctatccgga tattcttcca gcacctgaaa cgcggttttg cccggaatcg cggtggtcag
     3181 cagccacgca tcatccgggg tgcgaataaa atgtttaatg gtcggcagcg gcataaattc
     3241 ggtcagccag ttcagacgca ccatttcatc ggtcacatcg ttcgccacgc tgcctttgcc
     3301 atgtttcaga aacagttccg gcgcatccgg tttgccatac agacgataaa tggtcgcgcc
     3361 gctctgaccc acgttatcac gcgcccattt atagccatac agatccgcat ccatgttgct
     3421 gttcagacgc ggacggctac agctcgtttc acgctgaata tggctcataa caccccttgt
     3481 attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc
     3541 aatgtaacat cagagatttt gagacacaaa tttaaatcgt aattattggg gacccctgga
     3601 ttctcaccaa taaaaaacgc ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt
     3661 tctgaggtca ttactggatc tatcaacagg agtccaag
//
