LOCUS       sequence-199386-        3556 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of GFPmut2; strong ribosome binding site; 
            GFPmut2 ORF can be replaced with gene of interest by isothermal 
            assembly.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3556)
  AUTHORS   Hensel Z
  TITLE     A plasmid-based Escherichia coli gene expression system with 
            cell-to-cell variation below the extrinsic noise limit.
  JOURNAL   PLoS One. 2017 Oct 30;12(10):e0187259. doi: 
            10.1371/journal.pone.0187259. eCollection 2017.
  PUBMED    29084263
REFERENCE   2  (bases 1 to 3556)
  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: "PLoS One.";
            date: "2017-10-30"; volume: "12(10):e0187259. doi"; pages: " 
            10.1371/journal.pone.0187259. eCollection 2017"
COMMENT     Sequence Label: pZH514
FEATURES             Location/Qualifiers
     source          1..3556
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..19,20..25,26..42,43..48,49..74)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1..19
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    26..44
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             84..95
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             104..817
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(134..162)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     757..777
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      853..899
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(918..934)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(918..934)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(931..953)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    942..958
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(966..972,973..990,991..996))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1011..1032
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(1572..2117)
                     /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(1652..1671)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     regulatory      1671..1680
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(join(2308..3099,3100..3168))
                     /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     2931..2950
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3169..3273)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3341..3359
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3397..3419)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3519..3538
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tccctatcag tgatagagat tgacatccct atcagtgata gagatactga gcacatcagc
       61 aggacgcact gaccgaattc attaaagagg agaaaggtac cgcatgagta aaggagaaga
      121 acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa
      181 attttctgtc agtggagagg gtgaaggtga tgcaacatac ggaaaactta cccttaaatt
      241 tatttgcact actggaaaac tacctgttcc atggccaaca cttgtcacta ctttcgcgta
      301 tggtcttcaa tgctttgcga gatacccaga tcatatgaaa cagcatgact ttttcaagag
      361 tgccatgccc gaaggttatg tacaggaaag aactatattt ttcaaagatg acgggaacta
      421 caagacacgt gctgaagtca agtttgaagg tgataccctt gttaatagaa tcgagttaaa
      481 aggtattgat tttaaagaag atggaaacat tcttggacac aaattggaat acaactataa
      541 ctcacacaat gtatacatca tggcagacaa acaaaagaat ggaatcaaag ttaacttcaa
      601 aattagacac aacattgaag atggaagcgt tcaactagca gaccattatc aacaaaatac
      661 tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcca cacaatctgc
      721 cctttcgaaa gatcccaacg aaaagagaga ccacatggtc cttcttgagt ttgtaacagc
      781 tgctgggatt acacatggca tggatgaact atacaaataa taatctagcc atggtacgcg
      841 tgctagaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttata
      901 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
      961 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     1021 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1081 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     1141 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1201 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     1261 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggcgcgcta gcggagtgta
     1321 tactggctta ctatgttggc actgatgagg gtgtcagtga agtgcttcat gtggcaggag
     1381 aaaaaaggct gcaccggtgc gtcagcagaa tatgtgatac aggatatatt ccgcttcctc
     1441 gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg cttacgaacg
     1501 gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg
     1561 gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca
     1621 aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccatggcggc
     1681 tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc gctgttatgg
     1741 ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc gctccaagct
     1801 ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg
     1861 tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca ctggtaattg
     1921 atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt
     1981 ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta gctcagagaa
     2041 ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag attacgcgca
     2101 gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatataga tcttttctac
     2161 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     2221 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     2281 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     2341 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     2401 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     2461 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     2521 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     2581 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     2641 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     2701 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     2761 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     2821 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     2881 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     2941 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     3001 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     3061 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     3121 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     3181 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     3241 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     3301 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     3361 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3421 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     3481 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     3541 gagagtgcac catatg
//
