LOCUS       sequence-305577-        7158 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-GFP-TglG fusion in E. Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7158)
  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 7158)
  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-GFP-tglG
FEATURES             Location/Qualifiers
     source          1..7158
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     123..142
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     226..245
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        226..244
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    245..269
                     /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             284..306
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             325..342
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             352..369
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(382..384,385..387,388..1098)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(382..384,385..387,388..1098)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(427..448)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(688..707)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1035..1056
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2248..2265
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2318..2336)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2332..2379
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2416..2871
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2503..2522)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2713..2734
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2963..3778)
                     /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(2963..3778)
                     /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     3686..3705
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3770..3789
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3900..4488
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4389..4408
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4642..4659
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4674..4813
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4739..4758)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4899..4921
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4915..5106)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5915..6997)
                     /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     6959..6978
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6998..7075)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
       61 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
      121 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
      181 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
      241 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
      301 aggagatata ccatgggcag cagccatcat catcatcatc acagcagcgg cctggtgccg
      361 cgcggcagcc atatggctag catggtgagc aagggcgagg agctgttcac cggggtggtg
      421 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      481 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
      541 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
      601 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
      661 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
      721 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
      781 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
      841 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
      901 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
      961 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     1021 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1081 atggacgagc tgtacaagat gacacacgct tccgatgtgt ccgaggttat gaaacagact
     1141 tcaattgcgt cctgtctgcg caacgatatt gttgtcagcg agcgtgtgga ggcagacggc
     1201 cggttgcgct acaagcttta cgaccccagt aacgatatcg gttttgagtt gggtaaaacc
     1261 gagtatgcac tggcccgcct ttttgatggt gtatccacac acgaagagat ctcccgatac
     1321 gctgaaaccc ggcaccgcat caaggtcagt cagacgaaac tggtgcagtt tgaaagtcgc
     1381 ctattgagtc tgaatattct gtcgggggta gatggcgatc ttgaaaaact tcgtgatccc
     1441 gcgaccggta ttacctatgg gcctttgaag aaatacctgc tgatcaacct ggtcaagatg
     1501 aatccgcagc cgggcctgga ctatgtttac cccagcgtgc gctggatatg cagcgtaccg
     1561 ttttgtctgg cgatgcttgc ggcgtgtttg gtcagctacg tctacctggg gatgcacttc
     1621 ggcgcatttc aagaagatgt tctcgcggtg tatgtgcaca gcaatcaatg gctgtggtgg
     1681 cattttccgg tcatcctcag ttctatcgca ctgcacgagt tcgggcatgc cctggcctgt
     1741 cgacattaca aggttcaggt cagtgatttc gggatcggca tttatctgtt gctggccacc
     1801 gggtgggtgc ggccggttca ggaacaatgg agttcgttgc cgaggtatca gcgcatcgtc
     1861 accattttgg ccgggccact ggtcagcctt gtttacgcca ctgcaggcat tgtgatctgg
     1921 gctgcttacg gtgaagcgca aggtaacttg cttgcgctgt cgattgtgat gattgtggca
     1981 tcgttcatct ctctgattcc aaccttgttg ccaatgttca atggcgacac ttacctggcc
     2041 atcaccgaac tcatcaacga gccccggtta aggcagcgct ctttcaacta tttgaaagca
     2101 cgcttgcgcg gggcaccttg tgaaacgtca gtccggctcc aacgccttta tctgagcact
     2161 gtatcgatca cctgtcttgg ctgggtgttg gtgtggggct tcgtgctgca gacgcttttt
     2221 cgtgtatttc ataaccagtg actcgagcac caccaccacc accactgaga tccggctgct
     2281 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     2341 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     2401 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2461 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2521 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2581 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2641 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2701 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2761 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2821 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     2881 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2941 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     3001 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     3061 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     3121 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     3181 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     3241 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     3301 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     3361 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3421 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     3481 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3541 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3601 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     3661 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3721 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     3781 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     3841 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3901 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3961 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4021 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4081 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4141 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4201 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4261 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4321 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4381 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4441 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4501 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4561 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4621 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4681 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     4741 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4801 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4861 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4921 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     4981 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     5041 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     5101 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     5161 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     5221 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     5281 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     5341 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     5401 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     5461 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     5521 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     5581 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     5641 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     5701 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     5761 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     5821 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     5881 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5941 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     6001 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     6061 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     6121 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     6181 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     6241 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     6301 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     6361 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     6421 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     6481 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     6541 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     6601 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     6661 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     6721 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     6781 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     6841 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     6901 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     6961 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     7021 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     7081 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     7141 ccgttgagca ccgccgcc
//
