LOCUS       sequence-229516-        4675 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFPmut2 with low cell-to-cell variation and lactose 
            induction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4675)
  AUTHORS   Silva JPN, Lopes SV, Grilo DJ, Hensel Z
  TITLE     Plasmids for Independently Tunable, Low-Noise Expression of Two 
            Genes.
  JOURNAL   mSphere. 2019 May 29;4(3). pii: 4/3/e00340-19. doi: 
            10.1128/mSphere.00340-19.
  PUBMED    31142623
REFERENCE   2  (bases 1 to 4675)
  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: "mSphere."; 
            date: "2019-05-29"; volume: "4(3). pii: 4/3/e00340-19. doi"; pages: 
            " 10.1128/mSphere.00340-19"
COMMENT     Sequence Label: pJS102
FEATURES             Location/Qualifiers
     source          1..4675
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    215..231
                     /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)."
     RBS             273..284
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             293..1006
                     /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(323..351)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     946..966
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             1033..2115
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1052..1071)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     terminator      2160..2206
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2225..2241)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2225..2241)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2238..2260)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2249..2265
                     /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(2273..2279,2280..2297,2298..2303))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2318..2339
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2879..3424)
                     /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(2959..2978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3615..4406,4407..4475))
                     /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     4238..4257
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4476..4580)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4648..4666
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgat aaatgtgagc ggataacatt gacattgtga gcggataaca agatactgag
      241 cacatcagca ggacgcactg accgaattca ttaaagagga gaaaggtacc gcatgagtaa
      301 aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa
      361 tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac
      421 ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac
      481 tttcgcgtat ggtcttcaat gctttgcgag atacccagat catatgaaac agcatgactt
      541 tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga
      601 cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat
      661 cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata
      721 caactataac tcacacaatg tatacatcat ggcagacaaa caaaagaatg gaatcaaagt
      781 taacttcaaa attagacaca acattgaaga tggaagcgtt caactagcag accattatca
      841 acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac
      901 acaatctgcc ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt
      961 tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaataat aatctagcag
     1021 gaggatttca ccatgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     1081 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     1141 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     1201 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     1261 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     1321 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     1381 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     1441 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     1501 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     1561 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     1621 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     1681 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     1741 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     1801 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     1861 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     1921 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     1981 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     2041 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     2101 gaaagcgggc agtaataact gcagcccggg ggatcccatg gtacgcgtgc tagaggcatc
     2161 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttataagc ttggcgtaat
     2221 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     2281 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     2341 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     2401 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     2461 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2521 cggtaatacg gttatccaca gaatcagggg atagcgcagg aaagaacatg tgagcaaaag
     2581 gccagcaaaa ggccaggaac cgtaaaaagg cgcgctagcg gagtgtatac tggcttacta
     2641 tgttggcact gatgagggtg tcagtgaagt gcttcatgtg gcaggagaaa aaaggctgca
     2701 ccggtgcgtc agcagaatat gtgatacagg atatattccg cttcctcgct cactgactcg
     2761 ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg cggagatttc
     2821 ctggaagatg ccaggaagat acttaacagg gaagtgagag ggccgcggca aagccgtttt
     2881 tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc
     2941 gaaacccgac aggactataa agataccagg cgtttccccc tggcggctcc ctcgtgcgct
     3001 ctcctgttcc cgcctttcgg tttaccggtg tcattccgct gttatggccg cgtttgtctc
     3061 attccacgcc tgacactcag ttccgggtag gcagttcgct ccaagctgga ctgtatgcac
     3121 gaaccccccg ttcagtccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3181 ccggaaagac atgcaaaagc accactggca gcagccactg gtaattgatt tagaggagtt
     3241 agtcttgaag tcatgcgccg gttaaggcta aactgaaagg acaagttttg gtgactgcgc
     3301 tcctccaagc cagttacctc ggttcaaaga gttggtagct cagagaacct tcgaaaaacc
     3361 gccctgcaag gcggtttttt cgttttcaga gcaagagatt acgcgcagac caaaacgatc
     3421 tcaagaagat catcttatta atcagataaa atatagatct tttctacggg gtctgacgct
     3481 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     3541 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     3601 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     3661 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     3721 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     3781 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3841 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3901 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3961 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4021 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4081 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4141 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4201 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     4261 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4321 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     4381 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     4441 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     4501 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4561 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     4621 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
