LOCUS       sequence-305081-        7419 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-TglH and TglI in E. Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7419)
  AUTHORS   Ting CP, Funk MA, Halaby SL, Zhang Z, Gonen T, van der Donk WA
  TITLE     Use of a scaffold peptide in the biosynthesis of amino acid-derived 
            natural products.
  JOURNAL   Science. 2019 Jul 19;365(6450):280-284. doi: 
            10.1126/science.aau6232.
  PUBMED    31320540
REFERENCE   2  (bases 1 to 7419)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2019-07-19- 19"; volume: "365"; 
            issue: "6450"; pages: "280-284"
COMMENT     Sequence Label: pET15b-tglHI
FEATURES             Location/Qualifiers
     source          1..7419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     36..55
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     139..158
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        139..157
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    158..182
                     /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             197..219
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             238..255
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             265..282
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(2058..2076)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2072..2119
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2294..2299,2300..2316,2317..2322))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2349..2367)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2435..2539
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2540..2608,2609..3400)
                     /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(2758..2777)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3571..4159
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4060..4079
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4313..4330
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4345..4484
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4410..4429)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4570..4592
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4586..4777)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6089..7171)
                     /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     7133..7152
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7172..7249)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
       61 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
      121 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
      181 cccctctaga aataattttg tttaacttta agaaggagat ataccatggg cagcagccat
      241 catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatgat gcctgatttc
      301 gtcaagcccg cgcctatcgg agtgggtatc cagtacaacc ccgaaatact ggactggttt
      361 ccattcgagg atatccaggt ggatattctg gagattttgc tcgataacat catggcgccc
      421 atggatggcc cgcaaattat caagccttct gcccaggcaa tgattgagcg gctagggcaa
      481 aaatttacct tgctggctca ctccaattac ggctgcgatt ttggtttcag tgccctcgaa
      541 gaaacagcgg cggtgcagcg ccatgtacct ctggcaaaaa tgctcaacag cccttgggtg
      601 gccaaccatt gtttctatgg ggatcagtcc tggctggata tctggagcag tcccattcag
      661 ttctccgcag ccgaagtggc gcgttgtgcc gatcgcgcac agtcgttgca gacgctttat
      721 ggcatgccgc tggctcatga aaatgccgcc tactaccttg aatgcccagg cgccgaaatg
      781 cgtgaggccg agtttctggc aagactggtg cagcgctcag ggacttttct tcaccttgac
      841 ctgcacaaca tttacaccaa ccacctcaat ctgaaaggtt tcgatctcaa ggactacatg
      901 gacacgctgc cgctggacaa ggtgatttcg gtgcatctgg ccggaggaag ctggcacggc
      961 ggcctgtatc acgactggca tgacgcctgc gtgcccgagc cggtctggga cttgtacgaa
     1021 gacctgcttt cgcgtgcaca gccctctgct gtgattcttg agtatcaggg gcaggcgcat
     1081 catgcccaga ctaggatcat ggatgcgtcc gacgagtcga tgattgtcag ggatgtccag
     1141 cgtgctcagg caatctggag tcgttacaac aggcatccgc aggagcgtca gtatggaagc
     1201 tgaacaggta tggaaactgt ggcggcgcgt tctgcgcgat gagcgccttc aggcgcagtt
     1261 gttttcggct accgatgcca cgcactggtt gtcaggtttc agcgagtccg aaagcaagat
     1321 cctctcggtg tatgcgcagc agtttgaccg cgtcaaatgg ttcgtcgaga actatcagtt
     1381 ccggctggtg aactcttttt tgaacgcgct ggagaccggc gcgcccttgt cgttgcgggc
     1441 gttgctgcac ataaatgtgg acctgaatgc gcaatcgaag gccttcctgc gtgacaggca
     1501 gtggcgcgat tacgggccgc aggtctatac ctattgtgaa gatgtactgg gtttccttgc
     1561 cgaggcggat gagctgcagg gttatcctga aatccttgat ctgatgcggc tggagcgcga
     1621 atcggttcgc ctgtatcggg ggttggtcga tcctgagtcc ttgccggcag acaaccgtta
     1681 tcagcgcacc tcaatggctc gtctttacga gacacgtttt gccctgtctg gctggctcag
     1741 acagaaggat cagttgggcc tgacacgtct gcccgagagc accgagcatg tgctgatcta
     1801 ccttcccacg cttcaggctc gtcacaagtt cactttgatc aacgcgcagg ctgcccggct
     1861 ttacaactgc ctggagcaac ctcagtccgc tgccgggctg ttcatgctga tcaattcgga
     1921 ctcggcgtcc gtgccgggta gcgcagacct ggcgctgctc gacaggctgg aacagcttaa
     1981 cgcgataagg aagcctttgt agctcgagga tccggctgct aacaaagccc gaaaggaagc
     2041 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     2101 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatcccg caagaggccc
     2161 ggcagtaccg gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg
     2221 acgatgagcg cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt
     2281 gataaactac cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag
     2341 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     2401 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2461 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     2521 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     2581 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     2641 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     2701 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     2761 atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca
     2821 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     2881 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     2941 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3001 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3061 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3121 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3181 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3241 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3301 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     3361 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     3421 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     3481 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3541 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3601 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3661 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     3721 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3781 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3841 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3901 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3961 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4021 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4081 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4141 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4201 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4261 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4321 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4381 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4441 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     4501 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4561 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4621 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     4681 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     4741 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     4801 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     4861 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     4921 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     4981 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     5041 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     5101 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     5161 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     5221 ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc
     5281 ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca
     5341 cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt
     5401 gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg
     5461 gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc
     5521 cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag
     5581 agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat
     5641 ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc
     5701 catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc
     5761 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5821 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5881 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5941 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     6001 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     6061 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6121 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6181 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     6241 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     6301 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     6361 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     6421 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     6481 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     6541 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     6601 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     6661 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6721 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6781 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     6841 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     6901 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6961 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     7021 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     7081 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     7141 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     7201 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     7261 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     7321 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     7381 acggggcctg ccaccatacc cacgccgaaa caagcgctc
//
