LOCUS       sequence-267407-        8583 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG inducible system (gfpmut2 with Ppsank) for expression in B. 
            subtilis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8583)
  AUTHORS   Gonzalez LM, Mukhitov N, Voigt CA
  TITLE     Resilient living materials built by printing bacterial spores.
  JOURNAL   Nat Chem Biol. 2019 Dec 2. pii: 10.1038/s41589-019-0412-5. doi: 
            10.1038/s41589-019-0412-5.
  PUBMED    31792444
REFERENCE   2  (bases 1 to 8583)
  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: "Nat Chem 
            Biol. 2019 Dec 2. pii: 10.1038/s41589-019-0412-5. doi: 
            10.1038/s41589-019-0412-5."
COMMENT     Sequence Label: pLG163
FEATURES             Location/Qualifiers
     source          1..8583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      90..678
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     579..598
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      4187..4273
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4292..4319
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      4399..4433
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     protein_bind    4537..4553
                     /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)."
     CDS             4586..5296
                     /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="MKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(4613..4641)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5236..5256
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             5555..6637
                     /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
                     EACKTAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(5574..5593)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     primer_bind     complement(7451..7469)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7537..7641
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7642..7710,7711..8502)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7860..7879)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
       61 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
      121 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
      181 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
      241 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
      301 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
      361 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
      421 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
      481 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
      541 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
      601 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
      661 gggcggagcc tatggaaaaa cgccagcaac gcggcccgac ctcgagctgg atacttcccg
      721 tccgccaggg ggacatgccg gcgatgctga aggtcgcgcg cattcccgat gaagaggccg
      781 gttaccgcct gtttgaggat atagtaatct ttctaaatag ctttggattg gaggagtatg
      841 gccactaata ctaagttcag ctaataaaaa aatttgctaa agaactccag ctggatttca
      901 ctgatgagaa tatcgtcgga gataaatata ataattccac ggactataga ctatactagt
      961 atactccgtc tactgtacga tacacttccg ctcaggtcct tgtcctttaa cgaggattgt
     1021 tacgtacgct aatggcgtca aaacaaagac tctagaccta ggccttaaga tctgatcata
     1081 tgcatccgcg ggcccgggtt aacgcgtaat ccatggatca agagacagga tgaggatcgt
     1141 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggtgccctga atgaactgca
     1201 gaaagagctg gtagttggcg cactgttcga agaactgccg atgtccagta agattcttac
     1261 tatgctggtt gaaccggatg ctggtaaagc tacttgggtt gctgcttcta cttatggtac
     1321 cgatacaact actggtgagg aagttaaagg agctcttaaa gaaatccact tcagtaggtg
     1381 gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct
     1441 acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc
     1501 agcggtccaa tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc
     1561 tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg
     1621 ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gactaagaaa
     1681 atgccgtcaa atccgctcgc catgacttca ctaacgatgc ctttgaaaat cttcaagttc
     1741 ttttctacta attcaaggcg tgtctcacca ggtttttggt ttgctccggc gcaaatgcag
     1801 acaatatcag catccttgca gggtatgttt ctctttgatg tctttttgtt tgtgaagtat
     1861 ttcacattta tattgtgcaa cacttcacaa acttttgcaa gagaaaagtt ttgtctgatt
     1921 tatgaacaaa aaagaaacca tcattgatgg tttctttcgg taagtcccgt ctagccttgc
     1981 cctcaatggg gaagagaacc gcttaagccc gagtcattat ataaaccatt tagcacgtaa
     2041 tcaaagccag gctgattctg accgggcact tgggcgctgc cattattaaa aatcactttt
     2101 gcgttggttg tatccgtgtc cgcaggcagc gtcagcgtgt aaattccgtc tgcattttta
     2161 gtcattggtt ttccaggcca agatccggtc aattcaatta ctcggctccc atcatgttta
     2221 tagatataag catttacctg gctccaatga ttcggatttt gatagccgat ggttttggcc
     2281 gacgctggat ctcttttaac aaaactgtat ttctcggtcc tcgttacacc atcactgttc
     2341 gttcctttta acatgatggt gtatgttttg ccaaatggat ctccttttcc gattgtgaat
     2401 tgatctccat ccttaaacgc cgtctctggt ccattattga tttgataaac ggcttttgtt
     2461 gtattcgcat ctgcacgcaa ggtaatcgtc agttgatcat tgaaagaatg tgttacacct
     2521 gttttgtaat tctcaaggaa aacatgaggc gcttttgcaa tatcatcagg ataaagcaca
     2581 gctacagacc tggcattgat cgtgcctgtc agtttaccat cgttcacttg aaatgaaccc
     2641 gctccagctt tattgtcata cctgccatca ggcaattttg ttgccgtatt gatagagaca
     2701 gaggatgaac ctgcatttgc cagcacaacg ccatgtgagc cgcgctgatt cataaatatc
     2761 tggttgtttc cattcgggtt cgagagttcc tcaggctgtc cagccatcac attgtgaaat
     2821 ctattgaccg cagtgatagc ctgatcttca aataaagcac tcccgcgatc gcctatttgg
     2881 cttttccccg ggaacctcac accatttccg cctccctcag gtctggaaaa gaaaagaggc
     2941 gtactgcctg aacgagaagc tatcaccgcc cagcctaaac ggattccccc tatgcaaggg
     3001 tttattgttt tctaaaatct gattaccaat tagaatgaat atttcccaaa tattaaataa
     3061 taaaacaaaa aaattgaaaa aagtgtttcc accatttttt caattttttt ataatttttt
     3121 taatctgtta tttaaatagt ttatagttaa atttacattt tcattagtcc attcaatatt
     3181 ctctccaaga taactacgaa ctgctaacaa aattctctcc ctatgttcta atggagaaga
     3241 ttcagccact gcatttcccg caatatcttt tggtatgatt ttacccgtgt ccatagttaa
     3301 aatcatacgg cataaagtta atatagagtt ggtttcatca tcctgataat tatctattaa
     3361 ttcctctgac gaatccataa tggctcttct cacatcagaa aatggaatat caggtagtaa
     3421 ttcctctaag tcataatttc cgtatattct tttatttttt cgttttgctt ggtaaagcat
     3481 tatggttaaa tctgaattta attccttctg aggaatgtat ccttgttcat aaagctcttg
     3541 taaccattct ccataaataa attcttgttt gggaggatga ttccacggta ccatttcttg
     3601 ctgaataata attgttaatt caatatatcg taagttgctt ttatctccta ttttttttga
     3661 aataggtcta attttttgta taagtatttc tttactttga tctgtcaatg gttcagatac
     3721 gacgactaaa aagtcaagat cactatttgg ttttagtcca ctctcaactc ctgatccaaa
     3781 catgtaagta ccaataaggt tattttttaa atgtttccga agtatttttt tcactttatt
     3841 aatttgttcg tatgtattca aatatatcct cctcactatt ttgattagta cctattttat
     3901 atccatagtt gttaattaaa taaacttaat ttagtttatt tatagatttc attggcttct
     3961 aaatttttta tctagataat aattatttta gttaatttta ttctagatta tatatgatat
     4021 gatctttcat ttccataaaa ctaaagtaag tgtaaaccta ttcattgttt taaaaatatc
     4081 tcttgccagt cacgttacgt tattagttat agttattata acatgtattc acgaacgaaa
     4141 atcgccattc gccagctgca ggaattcgac ctctagcttg aggcatcaaa taaaacgaaa
     4201 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     4261 gagtaggaca aatccgccgc tctagctaag cagaaggcca tcctgacgga tggccttttt
     4321 gcgtttctac aaactcttgt taactctaga gctgcctgcc gcgtttcggt gatgaagatc
     4381 ttcccgatga ttaattaatt cagaacgctc ggttgccgcc gggcgttttt tatgcagcaa
     4441 tggcaagaac gttgctcgag ggtaaatgtg agcactcaca attcattttg caaaagttgt
     4501 tgactttatc tacaaggtgt ggcataatgt gtgtaattgt gagcggataa caattaagct
     4561 ttcggctgaa aggtggtgaa ctactatgaa aggagaagaa cttttcactg gagttgtccc
     4621 aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg
     4681 tgaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta ctggaaaact
     4741 acctgttcca tggccaacac ttgtcactac tttcgcgtat ggtcttcaat gctttgcgag
     4801 atacccagat catatgaaac agcatgactt tttcaagagt gccatgcccg aaggttatgt
     4861 acaggaaaga actatatttt tcaaagatga cgggaactac aagacacgtg ctgaagtcaa
     4921 gtttgaaggt gatacccttg ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga
     4981 tggaaacatt cttggacaca aattggaata caactataac tcacacaatg tatacatcat
     5041 ggcagacaaa caaaagaatg gaatcaaagt taacttcaaa attagacaca acattgaaga
     5101 tggaagcgtt caactagcag accattatca acaaaatact ccaattggcg atggccctgt
     5161 ccttttacca gacaaccatt acctgtccac acaatctaag ctttcgaaag atcccaacga
     5221 aaagagagac cacatggtcc ttcttgagtt tgtaacagct gctgggatta cacatggcat
     5281 ggatgaacta tacaaataag ctagccgcat gcaagctaat tcggtggaaa cgaggtcatc
     5341 atttccttcc gaaaaaacgg ttgcatttaa atcttacata tgtaatactt tcaaagacta
     5401 catttgtaag atttgatgtt tgagtcggct gaaagatcgt acgtaccaat tattgtttcg
     5461 tgattgttca agccataaca ctgtagggat agtggaaaga gtgcttcatc tggttacgat
     5521 caatcaaata ttcaaacgga gggagacgat tttgatgaaa ccagtaacgt tatacgatgt
     5581 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     5641 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     5701 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     5761 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     5821 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaaac
     5881 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     5941 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     6001 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     6061 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     6121 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     6181 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     6241 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     6301 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     6361 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     6421 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     6481 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     6541 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     6601 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     6661 aggatcctga gcgccggtcg ctaccattac cagttggtct ggtgtcaaaa ataataataa
     6721 ccgggcaggc catgtctgcc cgtatttcgc gtaaggaaat ccattatgta ctatttaatt
     6781 ctgcgtgaca tcccatcgat cagaccagtt tttaatttgt gtgtttccat gtgtccagtt
     6841 tggaatactc ttaacctcat tggaaatcgc ggcataatca ctggtggtat gattgatgac
     6901 cgcgtcaaca atgaccttta tgccatattc ttcagcggct gcacacattt ctttaaattc
     6961 ttgttcagta cctaagtaac ggttgccaat ttgatacgat gtcggctgat acagccagta
     7021 ccagttcgac atgcttttat ctccttgatt cccttccttt acttggttaa tcggagatgt
     7081 ctgaatggct gtatatcctg catcatgaat atccttcata ttgtgtttta acgtattgaa
     7141 cgaccaattc catgcatgaa gaatggttcc gcttttgatc gacggtgctg taagctcatt
     7201 cgatttgttc gccgtttcag cactcgcagc cgccggtcct gccagaacca aatgaaacag
     7261 caataaaaat ccagcgaata acggcagtaa agaggttttg aatcgttttg caaacattct
     7321 tgacactcct tatttgattt tttgaagact tacttcggag tcaaaaatcc ctcttacttc
     7381 attcttccgc ttcctccttt caaaccgatg tgaagactgg agaattttgt taattcttga
     7441 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     7501 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     7561 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     7621 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     7681 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     7741 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     7801 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     7861 ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata
     7921 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     7981 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     8041 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     8101 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     8161 gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact
     8221 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     8281 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     8341 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     8401 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     8461 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     8521 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     8581 atc
//
