LOCUS       sequence-414331-        6580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP-TolA expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6580)
  AUTHORS   Williams-Jones DP, Webby MN, Press CE, Gradon JM, Armstrong SR, 
            Szczepaniak J, Kleanthous C
  TITLE     Tunable force transduction through the Escherichia coli cell 
            envelope.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Nov 21;120(47):e2306707120. doi: 
            10.1073/pnas.2306707120. Epub 2023 Nov 16.
  PUBMED    37972066
REFERENCE   2  (bases 1 to 6580)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2023-11-21"; volume: "120(47):e2306707120. 
            doi"; pages: " 10.1073/pnas.2306707120. Epub 2023 Nov 16"
COMMENT     Sequence Label: pJS61
FEATURES             Location/Qualifiers
     source          1..6580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(96..974)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1001..1285
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1208..1227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1363..2076
                     /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"
     CDS             1363..2076
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1393..1421)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2016..2036
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2077..2106
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="ASMTGGQQMG"
     primer_bind     complement(3438..3455)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3608..3694
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3786..3813
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3832..3923
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3924..3992,3993..4784)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4142..4161)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4826..5281
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4913..4932)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5123..5144
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      5392..5980
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5881..5900
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6134..6151
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6317..6339
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6477..6496
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac
       61 tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca
      121 ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta
      181 aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata
      241 ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag
      301 cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag
      361 caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg
      421 tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct
      481 tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc
      541 ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc
      601 gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca
      661 tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga
      721 tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa
      781 acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata
      841 taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc
      901 ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt
      961 tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat
     1021 tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta
     1081 accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt
     1141 aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca
     1201 ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta
     1261 tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcaggag gaattcacca
     1321 tggtacccgg ggatcctcta gatttaagaa ggagatatac atatgagtaa aggagaagaa
     1381 cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa
     1441 ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt
     1501 atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac tttcgcgtat
     1561 ggtcttcaat gctttgcgag atacccagat catatgaaac agcatgactt tttcaagagt
     1621 gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga cgggaactac
     1681 aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa
     1741 ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata caactataac
     1801 tcacacaatg tatacatcat ggcagacaaa caaaagaatg gaatcaaagt taacttcaaa
     1861 attagacaca acattgaaga tggaagcgtt caactagcag accattatca acaaaatact
     1921 ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc
     1981 ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct
     2041 gctgggatta cacatggcat ggatgaacta tacaaagcta gcatgactgg tggacagcaa
     2101 atgggtcgcg gatccgtgtc aaaggcaacc gaacaaaacg acaagctcaa gcgggcgata
     2161 attatttcag cagtgctgca tgtcatctta tttgcggcgc tgatctggag ttcgttcgat
     2221 gagaatatag aagcttcagc cggaggcggc ggtggttcgt ccatcgacgc tgtcatggtt
     2281 gattcaggtg cggtagttga gcagtacaaa cgcatgcaaa gccaggaatc aagcgcgaag
     2341 cgttctgatg aacagcgcaa gatgaaggaa cagcaggctg ctgaagaact gcgtgagaaa
     2401 caagcggctg aacaggaacg cctgaagcaa cttgagaaag agcggttagc ggctcaggag
     2461 cagaaaaagc aggctgaaga agccgcaaaa caggccgagt taaagcagaa gcaagctgaa
     2521 gaggcggcag cgaaagcggc ggcagatgct aaagcgaagg ccgaagcaga tgctaaagct
     2581 gcggaagaag cagcgaagaa agcggctgca gacgcaaaga aaaaagcaga agcagaagcc
     2641 gccaaagccg cagccgaagc gcagaaaaaa gccgaggcag ccgctgcggc actgaagaag
     2701 aaagcggaag cggcagaagc agctgcagct gaagcaagaa agaaagcggc aactgaagct
     2761 gctgaaaaag ccaaagcaga agctgagaag aaagcggctg ctgaaaaggc tgcagctgat
     2821 aagaaagcgg cagcagagaa agctgcagcc gacaaaaaag cagcagaaaa agcggctgct
     2881 gaaaaggcag cagctgataa gaaagcagcg gcagaaaaag ccgccgcaga caaaaaagcg
     2941 gcagcggcaa aagctgcagc tgaaaaagcc gctgcagcaa aagcggccgc agaggcagat
     3001 gatattttcg gtgagctaag ctctggtaag aatgcaccga aaacgggggg aggggcgaaa
     3061 gggaacaatg cttcgcctgc cgggagtggt aatactaaaa acaatggcgc atcaggggcc
     3121 gatatcaata actatgccgg gcagattaaa tctgctatcg aaagtaagtt ctatgacgca
     3181 tcgtcctatg caggcaaaac ctgtacgctg cgcataaaac tggcacccga tggtatgtta
     3241 ctggatatca aacctgaagg tggcgatccc gcactttgtc aggctgcgtt ggcagcagct
     3301 aaacttgcga agatcccgaa accaccaagc caggcagtat atgaagtgtt caaaaacgcg
     3361 ccattggact tcaaaccgta agtcgacctg caggcatgca agcttggctg ttttggcgga
     3421 tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg tctgataaaa
     3481 cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa
     3541 gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt agggaactgc
     3601 caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     3661 tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc
     3721 gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat
     3781 taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc ttttgtttat
     3841 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3901 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3961 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     4021 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     4081 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     4141 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     4201 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     4261 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4321 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4381 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4441 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     4501 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4561 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4621 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4681 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4741 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4801 actcatatat actttagatt gatttacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4861 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     4921 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4981 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5041 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5101 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     5161 atctcgggct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     5221 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5281 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5341 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5401 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5461 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5521 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5581 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5641 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5701 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5761 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5821 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5881 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5941 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6001 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6061 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     6121 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     6181 tctccttacg catctgtgcg gtatttcaca ccgcataggg tcatggctgc gccccgacac
     6241 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     6301 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     6361 cgcgcgaggc agaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     6421 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     6481 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     6541 cgtccggcgt agaggatctg ctcatgtttg acagcttatc
//
