LOCUS       sequence-305586-        6207 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-TglF in E. Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6207)
  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 6207)
  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: pET28b-tglF
FEATURES             Location/Qualifiers
     source          1..6207
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     53..72
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     156..175
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        156..174
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    175..199
                     /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             214..236
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             255..272
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             282..299
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             927..938
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1227..1244
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1297..1315)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1311..1358
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1395..1850
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1482..1501)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1692..1713
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1942..2757)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1942..2757)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2665..2684
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2749..2768
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2879..3467
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3368..3387
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3621..3638
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3653..3792
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3718..3737)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3878..3900
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3894..4085)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4894..5976)
                     /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     5938..5957
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5977..6054)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
       61 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
      121 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt
      181 gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata
      241 ccatgggcag cagccatcat catcatcatc acagcagcgg cctggtgccg cgcggcagcc
      301 atatgaacga ttgggctatc agccattacc tggatgtgtt gcacatggac agcggttatc
      361 aagccgttgc actggactgc gaactctgcg gcagtgaaca ccatgaggtg ttgctgccac
      421 gtatctgcgg tcctgaaccc cacggcgttg atttgcctgt gttggggtgt caggcgtgcg
      481 gccatatttt tcaaaaatat cgttttgatg cgcagtttta caaagactac tacgacaagt
      541 tttatcgctt gaaactgttc ggcaacagcg cgccggaaaa agcgtttttt ctcgaccaga
      601 ttcgtcgtgg agaatacctg cgtcagagcc tgcgcgaata cctgccagag agtggtcgcc
      661 tgctggatgt cggatgttca gccggtggcc tgatgatccc ttttgccaag aggggctggt
      721 ctgtcaccgg caatgaccct gatcgcgctt atgtcgaata cggaaaacag atcggcctga
      781 atatcgagct gacagcggcc gaagaaatgg acgccgcagg cgatgtcgat ctgattatca
      841 tcaatggctc gctggaacat atgtacgacg tgcattgcgt catgcagcgc tgtcggcagg
      901 cgtcgtcgga aaacggtctg ttattgattg aaggccgcgc cctgggtcac ggcctggagc
      961 agggcatgct gacccacaat catcggcgtt atctcagcgg caccagcatc gaactgctga
     1021 tgcgcatgca cggctgggag ccgctgctga tcaccgagca accgttatgc gggcctactc
     1081 gacctggcgc ggttttcgtc cttggtcgat gcgcagcccc gtcggatgat gtgcagctcg
     1141 aacgtgtcaa ggcgcaaggg cgtgagcgcc tggacagtta ctaccgggag cgcttccggg
     1201 cgctggtgtt gccatcatag ctcgagcacc accaccacca ccactgagat ccggctgcta
     1261 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     1321 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     1381 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1441 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1501 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     1561 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     1621 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     1681 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     1741 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     1801 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     1861 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1921 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     1981 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     2041 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     2101 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     2161 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     2221 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     2281 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     2341 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     2401 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     2461 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     2521 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     2581 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     2641 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     2701 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     2761 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     2821 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2881 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2941 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3001 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3061 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3121 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3181 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3241 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3301 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3361 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3421 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3481 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3541 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3601 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3661 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     3721 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     3781 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3841 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3901 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     3961 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     4021 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     4081 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     4141 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     4201 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     4261 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     4321 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     4381 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     4441 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     4501 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     4561 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     4621 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     4681 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     4741 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     4801 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     4861 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4921 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4981 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     5041 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     5101 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     5161 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     5221 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     5281 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     5341 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     5401 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     5461 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     5521 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     5581 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     5641 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     5701 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     5761 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     5821 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     5881 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     5941 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     6001 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     6061 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     6121 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     6181 cggccacggg gcctgccacc ataccca
//
