LOCUS       sequence-82628-e        6236 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses alpha hemolysin under the control of theophylline 
            riboswitch.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6236)
  AUTHORS   Lentini R, Santero SP, Chizzolini F, Cecchi D, Fontana J, 
            Marchioretto M, Del Bianco C, Terrell JL, Spencer AC, Martini L, 
            Forlin M, Assfalg M, Dalla Serra M, Bentley WE, Mansy SS
  TITLE     Integrating artificial with natural cells to translate chemical 
            messages that direct E. coli behaviour.
  JOURNAL   Nat Commun. 2014 May 30;5:4012. doi: 10.1038/ncomms5012.
  PUBMED    24874202
REFERENCE   2  (bases 1 to 6236)
  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 
            Commun."; date: "2014-05-30"; volume: "5:4012. doi"; pages: " 
            10.1038/ncomms5012"
COMMENT     Sequence Label: JF001A
FEATURES             Location/Qualifiers
     source          1..6236
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..23
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4..22
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             967..984
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1037..1055)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1051..1098
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1135..1590
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1222..1241)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1432..1453
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1617..1721
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1722..1790,1791..2582)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1940..1959)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2753..3341
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3242..3261
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3495..3512
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3527..3669
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3595..3614)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3755..3777
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3771..3962)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4771..5853)
                     /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     5815..5834
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5854..5931)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6137..6156
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aattaatacg actcactata gggtgatacc agcatcgtct tgatgccctt ggcagcaccc
       61 tgctaaggta acaacaagat ggattctgat atcaatatca aaaccggcac caccgatatc
      121 ggctccaata ccaccgttaa aaccggtgat ctggtgacct atgataaaga aaacggtatg
      181 cataaaaaag tgttttactc gtttattgac gataaaaacc ataacaaaaa actgctggtc
      241 atccgcacca aaggcaccat tgcgggtcaa taccgtgtgt actccgaaga aggtgcgaac
      301 aaaagcggtc tggcttggcc gtctgccttt aaagtgcagc tgcaactgcc ggataatgaa
      361 gtggcgcaga tttcagatta ttatccgcgt aatagcatcg ataccaaaga atatatgagt
      421 accctgacct atggttttaa tggcaatgtt accggtgatg atacgggtaa aattggcggt
      481 ctgattggcg ccaatgtgtc cattggtcat acgctgaaat acgtgcaacc ggatttcaaa
      541 accattctgg aaagtccgac cgataaaaaa gtgggttgga aagttatctt caacaacatg
      601 gtgaatcaga actggggtcc gtacgatcgc gattcctgga atccggttta tggcaatcag
      661 ctgtttatga aaacccgcaa cggtagtatg aaagcggcgg ataattttct ggacccgaac
      721 aaagcctcaa gcctgctgtc cagcggtttt agcccggatt ttgccacggt tattaccatg
      781 gatcgcaaag ccagcaaaca gcagaccaac attgatgtga tctacgaacg tgtgcgtgat
      841 gattatcaac tgcattggac ctcaaccaat tggaaaggca ccaataccaa agataaatgg
      901 acggatcgca gttcagaacg ctacaaaatt gattgggaaa aagaagaaat gaccaactaa
      961 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     1021 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1081 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     1141 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1201 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1261 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1321 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1381 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1441 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1501 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1561 attttaacaa aatattaacg tttacaattt caggtggcac ttttcgggga aatgtgcgcg
     1621 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1681 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1741 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1801 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1861 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1921 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     1981 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2041 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2101 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2161 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2221 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     2281 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2341 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2401 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2461 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2521 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2581 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2641 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2701 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2761 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2821 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2881 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2941 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3001 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3061 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3121 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3181 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3241 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3301 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3361 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3421 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3481 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3541 ttctccttac gcatctgtgc ggtatttcac accgcatata tggtgcactc tcagtacaat
     3601 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     3661 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3721 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3781 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     3841 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     3901 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     3961 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     4021 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     4081 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     4141 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     4201 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     4261 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     4321 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     4381 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     4441 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     4501 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     4561 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     4621 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     4681 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     4741 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4801 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4861 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4921 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4981 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     5041 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     5101 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     5161 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     5221 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     5281 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     5341 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     5401 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5461 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     5521 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5581 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     5641 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     5701 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     5761 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5821 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5881 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5941 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     6001 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     6061 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     6121 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     6181 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcga
//
