LOCUS       sequence-51016-e        2965 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2965)
  AUTHORS   Huang DC, Holtz WJ, Maharbiz MM
  TITLE     A genetic bistable switch utilizing nonlinear protein degradation.
  JOURNAL   J Biol Eng. 2012 Jul 9;6(1):9.
  PUBMED    22776405
REFERENCE   2  (bases 1 to 2965)
  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: "J Biol 
            Eng."; date: "2012-07-9"; volume: "6(1)"; pages: "9"
COMMENT     Sequence Label: PRM-GFP
FEATURES             Location/Qualifiers
     source          1..2965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             98..109
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(116..118,119..829)
                     /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             116..829
                     /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(146..174)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     769..789
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             830..862
                     /codon_start=1
                     /product="C-terminal peptide that mediates degradation in 
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
                     /label=ssrA tag (LVA)
                     /note="mutant LVA variant that confers accelerated 
                     degradation under some conditions (Andersen et al., 1998)"
                     /translation="AANDENYALVA"
     terminator      901..972
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      988..1015
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(1043..1060)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1177..1722)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(1257..1276)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1836..1930)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(1960..2751,2752..2820))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2583..2602
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2821..2925)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcaaccatta tcaccgccag aggtaaaata gtcaacacgc acggtgttag atatttataa
       61 atagtggtga tagatttaac gtggatctca attcattaaa gaggagaaag gtaccatgag
      121 caaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt
      181 taatgggcac aaattttctg tccgtggaga gggtgaaggt gatgctacaa acggaaaact
      241 cacccttaaa tttatttgca ctactggaaa actacctgtt ccgtggccaa cacttgtcac
      301 tactctgacc tatggtgttc aatgcttttc ccgttatccg gatcacatga aacggcatga
      361 ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa cgcactatat ctttcaaaga
      421 tgacgggacc tacaagacgc gtgctgaagt caagtttgaa ggtgataccc ttgttaatcg
      481 tatcgagtta aagggtattg attttaaaga agatggaaac attcttggac acaaacttga
      541 gtacaacttt aactcacaca atgtatacat cacggcagac aaacaaaaga atggaatcaa
      601 agctaacttc aaaattcgcc acaacgttga agatggttcc gttcaactag cagaccatta
      661 tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc
      721 gacacaatct gtcctttcga aagatcccaa cgaaaagcgt gaccacatgg tccttcttga
      781 gtttgtaact gctgctggga ttacacatgg catggatgag ctctacaaag ctgcaaacga
      841 cgaaaactac gctttagtag cttaataagg atcctaactc gagtaaggat ctccaggcat
      901 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
      961 gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat
     1021 acctagggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc
     1081 ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac
     1141 ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga
     1201 caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag
     1261 ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt
     1321 taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt
     1381 ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc
     1441 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac
     1501 cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt
     1561 taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg
     1621 ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg
     1681 ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat
     1741 cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
     1801 agccccatac gatataagtt gttactagtg cttggattct caccaataaa aaacgcccgg
     1861 cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc
     1921 aacaggagtc caagcgagct cgtaaacttg gtctgacagt taccaatgct taatcagtga
     1981 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     2041 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     2101 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     2161 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     2221 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     2281 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     2341 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     2401 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     2461 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     2521 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     2581 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     2641 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     2701 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     2761 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     2821 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     2881 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     2941 agtgccacct gaattcatga gatct
//
