LOCUS       sequence-111980-        3037 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFPuv Expression, Low Phosphate Dependent E. coli Expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3037)
  AUTHORS   Menacho-Melgar R, Ye Z, Moreb EA, Yang T, Efromson JP, Decker JS, 
            Wang R, Lynch MD
  TITLE     Scalable, two-stage, autoinduction of recombinant protein expression
            in E. coli utilizing phosphate depletion.
  JOURNAL   Biotechnol Bioeng. 2020 May 22. doi: 10.1002/bit.27440.
  PUBMED    32441815
REFERENCE   2  (bases 1 to 3037)
  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: "Biotechnol 
            Bioeng. 2020 May 22. doi: 10.1002/bit.27440."
COMMENT     Sequence Label: pSMART-GFPuv
FEATURES             Location/Qualifiers
     source          1..3037
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      12..83
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      99..126
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             join(408..410,411..413,414..1127)
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(441..469)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1064..1084
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1314..1345
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        1346..1448
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1449..2264
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1522..1541)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2288..2315
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(2327..2914)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2407..2426)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2936..2965)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
ORIGIN
        1 tgcccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
       61 gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt
      121 tctgcgttta tacacagcta acaccacgtc gtccctatct gctgccctag gtctatgagt
      181 ggttgctgga taacgtgcgt aattgtgctg atctcttata tagctgctct cattatctct
      241 ctaccctgaa gtgactctct cacctgtaaa aataatatct cacaggctta atagtttctt
      301 aatacaaagc ctgtaaaacg tcaggataac ttctatattc agggagacca caacggtttc
      361 cctctacaaa taattttgtt taactttcgt gtgtaggagg ataatctatg gctagcaaag
      421 gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg
      481 ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc tacatacgga aagcttaccc
      541 ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt gtcactactt
      601 tctcttatgg tgttcaatgc ttttcccgtt atccggatca tatgaaacgg catgactttt
      661 tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc aaagatgacg
      721 ggaactacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt aatcgtatcg
      781 agttaaaagg tattgatttt aaagaagatg gaaacattct cggacacaaa ctcgagtaca
      841 actataactc acacaatgta tacatcacgg cagacaaaca aaagaatgga atcaaagcta
      901 acttcaaaat tcgccacaac attgaagatg gatccgttca actagcagac cattatcaac
      961 aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtcgacac
     1021 aatctgccct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt cttgagtttg
     1081 taactgctgc tgggattaca catggcatgg atgagctcta caaataatga ggatccccgg
     1141 cttatcggtc agtttcacct gatttacgta aaaacccgct tcggcgggtt tttgcttttg
     1201 gaggggcaga aagatgaatg actgtccacg acgctatacc caaaagaaag acgaattctc
     1261 tagatatcgc tcaatactga ccatttaaat catacctgac ctccatagca gaaagtcaaa
     1321 agcctccgac cggaggcttt tgacttgatc ggcacgtaag aggttccaac tttcaccata
     1381 atgaaataag atcactaccg ggcgtatttt ttgagttatc gagattttca ggagctaagg
     1441 aagctaaaat gagccatatt caacgggaaa cgtcttgctc gaggccgcga ttaaattcca
     1501 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     1561 cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     1621 aaggtagcgt tgccaatgat gttacagatg agatggtcag gctaaactgg ctgacggaat
     1681 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     1741 ccactgcgat cccagggaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     1801 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     1861 attgtccttt taacggcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     1921 acggtttggt tggtgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     1981 tctggaaaga aatgcataag cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     2041 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     2101 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     2161 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     2221 tgaataaatt gcagtttcac ttgatgctcg atgagttttt ctaatgaggg cccaaatgta
     2281 atcacctggc tcaccttcgg gtgggccttt ctgcgttgct ggcgtttttc cataggctcc
     2341 gcccccctga cgagcatcac aaaaatcgat gctcaagtca gaggtggcga aacccgacag
     2401 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2461 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     2521 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     2581 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     2641 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     2701 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     2761 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttacctcg gaaaaagagt
     2821 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     2881 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgattt tctaccgaag
     2941 aaaggcccac ccgtgaaggt gagccagtga gttgattgca gtccagttac gctggagtct
     3001 gaggctcgtc ctgaatgata tcaagcttga attcgtt
//
