LOCUS       sequence-67471-e        9965 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses ads with trxA tag under Pgrac promoter (inducible by IPTG)
            in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9965)
  AUTHORS   Zhou K, Zou R, Zhang C, Stephanopoulos G, Too HP
  TITLE     Optimization of amorphadiene synthesis in Bacillus subtilis via 
            transcriptional, translational, and media modulation.
  JOURNAL   Biotechnol Bioeng. 2013 Mar 8. doi: 10.1002/bit.24900.
  PUBMED    23483530
REFERENCE   2  (bases 1 to 9965)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Biotechnol 
            Bioeng. 2013 Mar 8. doi: 10.1002/bit.24900."
COMMENT     Sequence Label: pHT-trxA.ads
FEATURES             Location/Qualifiers
     source          1..9965
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1223..1245
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1237..1254
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1238..1254
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(1297..2379)
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2341..2360
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    2763..2787
                     /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             2809..3135
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             4798..4815
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        8042..8146
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8147..8215,8216..9007)
                     /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     complement(8365..8384)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9178..9766
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9667..9686
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9920..9937
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ttaagttatt ggtatgactg gttttaagcg caaaaaaagt tgctttttcg tacctattaa
       61 tgtatcgttt tagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa
      121 gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat
      181 aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt
      241 tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta ttattgatat
      301 ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag
      361 gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt
      421 ataaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt
      481 tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc
      541 cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga
      601 aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa
      661 tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct
      721 cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa
      781 tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc
      841 ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat
      901 ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaagac cacattaaaa
      961 aatgtggtct tttgtgtttt tttaaaggat ttgagcgtag cgaaaaatcc ttttctttct
     1021 tatcttgata ataagggtaa ctattgccga tcgtccattc cgacagcatc gccagtcact
     1081 atggcgtgct gctagcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     1141 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     1201 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc
     1261 gagctcaggc cttaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     1321 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     1381 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     1441 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     1501 aaaatcctgt ttgatggtgg ttgacggcgg gatataacat gagctgtctt cggtatcgtc
     1561 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     1621 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     1681 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     1741 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     1801 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     1861 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     1921 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     1981 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     2041 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     2101 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     2161 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     2221 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     2281 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     2341 ggcatactct gcgacatcgt ataacgttac tggtttcatc aaaatcgtct ccctccgttt
     2401 gaatatttga ttgatcgtaa ccagatgaag cactctttcc actatcccta cagtgttatg
     2461 gcttgaacaa tcacgaaaca ataattggta cgtacgatct ttcagccgac tcaaacatca
     2521 aatcttacaa atgtagtctt tgaaagtatt acatatgtaa gatttaaatg caaccgtttt
     2581 ttcggaagga aatgatgacc tcgtttccac cggaattagc ttggtaccag ctattgtaac
     2641 ataatcggta cgggggtgaa aaagctaacg gaaaagggag cggaaaagaa tgatgtaagc
     2701 gtgaaaaatt ttttatctta tcacttgaaa ttggaaggga gattctttat tataagaatt
     2761 gtggaattgt gagcggataa caattcccaa ttaaaggagg aaggatccat gagcgataaa
     2821 attattcacc tgactgacga cagttttgac acggatgtac tcaaagcgga cggggcgatc
     2881 ctcgtcgatt tctgggcaga gtggtgcggt ccgtgcaaaa tgatcgcccc gattctggat
     2941 gaaatcgctg acgaatatca gggcaaactg accgttgcaa aactgaacat cgatcaaaac
     3001 cctggcactg cgccgaaata tggcatccgt ggtatcccga ctctgctgct gttcaaaaac
     3061 ggtgaagtgg cggcaaccaa agtgggtgca ctgtctaaag gtcagttgaa agagttcctc
     3121 gacgctaacc tggcgggctc aggcggcggc ggctcagagc tctctctgac tgaggaaaaa
     3181 ccaatccgtc cgatcgcaaa ctttccgccg agcatctggg gtgatcagtt cctgatctac
     3241 cagaagcagg tcgagcaagg cgtagaacag atcgtgaacg acctgaaaaa ggaggtacgt
     3301 cagctgctga aagaggctct ggacatcccg atgaaacatg cgaacctgct gaagctgatt
     3361 gatgagattc agcgcctggg tatcccatac cacttcgaac gtgaaattga tcacgcgctg
     3421 cagtgtattt atgaaacgta tggtgataac tggaacggtg accgtagctc cctgtggttc
     3481 cgtctgatgc gtaagcaggg ttattacgtg acctgcgacg tcttcaacaa ctacaaagac
     3541 aagaacggcg cgtttaaaca gagcctggcg aatgacgttg aaggcctgct ggagctgtat
     3601 gaagcaacct ctatgcgtgt tccgggcgaa atcatcctgg aagatgctct gggtttcacc
     3661 cgttctcgcc tgtctatcat gaccaaggac gcattttcca ctaacccggc cctgttcacc
     3721 gaaatccagc gtgcgctgaa acagcctctg tggaagcgtc tgccgcgtat cgaggcggca
     3781 cagtacatcc cgttctatca gcaacaggat tcccacaaca aaaccctgct gaaactggcg
     3841 aaactggaat ttaacctgct gcaatccctg cacaaagaag aactgtctca tgtttgcaaa
     3901 tggtggaaag ccttcgacat caagaaaaac gcgccgtgcc tgcgcgaccg tatcgtagaa
     3961 tgctatttct ggggcctggg ctccggttat gagcctcaat actctcgtgc tcgcgtattc
     4021 ttcaccaaag cggtagctgt gatcaccctg atcgacgata cctacgatgc ttacggtacg
     4081 tacgaagaac tgaagatttt taccgaggct gttgaacgct ggtctatcac ttgcctggat
     4141 accctgccgg aatacatgaa accgatctat aaactgttca tggatactta taccgaaatg
     4201 gaagagttcc tggcaaaaga aggtcgcact gatctgttca actgtggcaa ggagtttgtg
     4261 aaagaattcg tccgcaacct gatggtggag gcgaagtggg caaacgaggg tcacatcccg
     4321 accaccgaag aacacgaccc ggtagttatc atcaccggtg gcgcaaacct gctgactacc
     4381 acttgctacc tgggtatgtc cgacattttt acgaaagaaa gcgttgaatg ggcagtgtct
     4441 gcgcctccgc tgttccgtta ctctggtatc ctgggccgtc gcctgaacga tctgatgacc
     4501 cataaagccg agcaggaacg taaacactcc agcagctctc tggaatctta catgaaagaa
     4561 tacaacgtta acgaagagta tgcgcagacc ctgatctaca aagaggttga ggatgtatgg
     4621 aaagacatca accgtgagta cctgactact aaaaacatcc cacgcccgct gctgatggca
     4681 gtgatttatc tgtgccagtt tctggaagtg cagtatgcgg gcaaagataa cttcacccgt
     4741 atgggtgacg aatacaaaca cctgatcaag tccctgctgg tttacccaat gtccatccat
     4801 catcatcacc atcactgatc tagagtcgac gtccccgggg cagcccgcct aatgagcggg
     4861 cttttttcac gtcacgcgtc catggagatc tttgtctgca actgaaaagt ttatacctta
     4921 cctggaacaa atggttgaaa catacgaggc taatatcggc ttattaggaa tagtccctgt
     4981 actaataaaa tcaggtggat cagttgatca gtatattttg gacgaagctc ggaaagaatt
     5041 tggagatgac ttgcttaatt ccacaattaa attaagggaa agaataaagc gatttgatgt
     5101 tcaaggaatc acggaagaag atactcatga taaagaagct ctaaaactat tcaataacct
     5161 tacaatggaa ttgatcgaaa gggtggaagg ttaatggtac gaaaattagg ggatctacct
     5221 agaaagccac aaggcgatag gtcaagctta aagaaccctt acatggatct tacagattct
     5281 gaaagtaaag aaacaacaga ggttaaacaa acagaaccaa aaagaaaaaa agcattgttg
     5341 aaaacaatga aagttgatgt ttcaatccat aataagatta aatcgctgca cgaaattctg
     5401 gcagcatccg aagggaattc atattactta gaggatacta ttgagagagc tattgataag
     5461 atggttgaga cattacctga gagccaaaaa actttttatg aatatgaatt aaaaaaaaga
     5521 accaacaaag gctgagacag actccaaacg agtctgtttt tttaaaaaaa atattaggag
     5581 cattgaatat atattagaga attaagaaag acatgggaat aaaaatattt taaatccagt
     5641 aaaaatatga taagattatt tcagaatatg aagaactctg tttgtttttg atgaaaaaac
     5701 aaacaaaaaa aatccaccta acggaatctc aatttaacta acagcggcca aactgagaag
     5761 ttaaatttga gaaggggaaa aggcggattt atacttgtat ttaactatct ccattttaac
     5821 attttattaa accccataca agtgaaaatc ctcttttaca ctgttccttt aggtgatcgc
     5881 ggagggacat tatgagtgaa gtaaacctaa aaggaaatac agatgaatta gtgtattatc
     5941 gacagcaaac cactggaaat aaaatcgcca ggaagagaat caaaaaaggg aaagaagaag
     6001 tttattatgt tgctgaaacg gaagagaaga tatggacaga agagcaaata aaaaactttt
     6061 ctttagacaa atttggtacg catatacctt acatagaagg tcattataca atcttaaata
     6121 attacttctt tgatttttgg ggctattttt taggtgctga aggaattgcg ctctatgctc
     6181 acctaactcg ttatgcatac ggcagcaaag acttttgctt tcctagtcta caaacaatcg
     6241 ctaaaaaaat ggacaagact cctgttacag ttagaggcta cttgaaactg cttgaaaggt
     6301 acggttttat ttggaaggta aacgtccgta ataaaaccaa ggataacaca gaggaatccc
     6361 cgatttttaa gattagacgt aaggttcctt tgctttcaga agaactttta aatggaaacc
     6421 ctaatattga aattccagat gacgaggaag cacatgtaaa gaaggcttta aaaaaggaaa
     6481 aagagggtct tccaaaggtt ttgaaaaaag agcacgatga atttgttaaa aaaatgatgg
     6541 atgagtcaga aacaattaat attccagagg ccttacaata tgacacaatg tatgaagata
     6601 tactcagtaa aggagaaatt cgaaaagaaa tcaaaaaaca aatacctaat cctacaacat
     6661 cttttgagag tatatcaatg acaactgaag aggaaaaagt cgacagtact ttaaaaagcg
     6721 aaatgcaaaa tcgtgtctct aagccttctt ttgatacctg gtttaaaaac actaagatca
     6781 aaattgaaaa taaaaattgt ttattacttg taccgagtga atttgcattt gaatggatta
     6841 agaaaagata tttagaaaca attaaaacag tccttgaaga agctggatat gttttcgaaa
     6901 aaatcgaact aagaaaagtg caataaactg ctgaagtatt tcagcagttt tttttattta
     6961 gaaatagtga aaaaaatata atcagggagg tatcaatatt taatgagtac tgatttaaat
     7021 ttatttagac tggaattaat aattaacacg tagactaatt aaaatttaat gagggataaa
     7081 gaggatacaa aaatattaat ttcaatccct attaaatttt aacaaggggg ggattaaaat
     7141 ttaattagag gtttatccac aagaaaagac cctaataaaa tttttactag ggttataaca
     7201 ctgattaatt tcttaatggg ggagggatta aaatttaatg acaaagaaaa caatctttta
     7261 agaaaagctt ttaaaagata ataataaaaa gagctttgcg attaagcaaa actctttact
     7321 ttttcattga cattatcaaa ttcatcgatt tcaaattgtt gttgtatcat aaagttaatt
     7381 ctgttttgca caaccttttc aggaatataa aacacatctg aggcttgttt tataaactca
     7441 gggtcgctaa agtcaatgta acgtagcata tgatatggta tagcttccac ccaagttagc
     7501 ctttctgctt cttctgaatg tttttcatat acttccatgg gtatctctaa atgattttcc
     7561 tcatgtagca aggtatgagc aaaaagttta tggaattgat agttcctctc tttttcttca
     7621 acttttttat ctaaaacaaa cactttaaca tctgagtcaa tgtaagcata agatgttttt
     7681 ccagtcataa tttcaatccc aaatctttta gacagaaatt ctggacgtaa atcttttggt
     7741 gaaagaattt ttttatgtag caatatatcc gatacagcac cttctaaaag cgttggtgaa
     7801 tagggcattt tacctatctc ctctcatttt gtggaataaa aatagtcata ttcgtccatc
     7861 tacctatcct attatcgaac agttgaactt tttaatcaag gatcagtcct ttttttcatt
     7921 attcttaaac tgtgctctta actttaacaa ctcgatttgt ttttccagat ctcgagggta
     7981 actagcctcg ccgatcccgc aagaggcccg gcagtcaggt ggcacttttc ggggaaatgt
     8041 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     8101 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     8161 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     8221 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     8281 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     8341 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     8401 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     8461 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     8521 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     8581 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     8641 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     8701 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     8761 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     8821 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     8881 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     8941 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     9001 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     9061 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     9121 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     9181 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     9241 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     9301 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     9361 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     9421 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     9481 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     9541 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     9601 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     9661 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     9721 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     9781 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     9841 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     9901 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     9961 catgc
//
