LOCUS       sequence-305604-        8034 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-AmmB2 in E. Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8034)
  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 8034)
  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: pET28a-ammB2
FEATURES             Location/Qualifiers
     source          1..8034
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     34..53
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     137..156
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        137..155
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    156..180
                     /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             195..217
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             236..253
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             263..280
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3035..3052
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3105..3123)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3119..3166
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3203..3658
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3290..3309)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3500..3521
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3750..4565)
                     /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(3750..4565)
                     /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     4473..4492
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4557..4576
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4687..5275
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5176..5195
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5429..5446
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5461..5600
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5526..5545)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5686..5708
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5702..5893)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6702..7784)
                     /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     7746..7765
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7785..7862)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
       61 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
      121 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
      181 cctctagaaa taattttgtt taactttaag aaggagatat accatgggca gcagccatca
      241 tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc catatgatgt caactcctgc
      301 atggcatgtt cttcctacag tcgttgtgcg tagtgcaggt ttcccatggc ggttagtaga
      361 tagacttgga caccgtaaga gccgccgctt agctggagag ttggttgcac tgagaactga
      421 agcagcagaa ttagcagcta cagctgcacc aacaggtaga ttagctcgtt ctacagcagc
      481 tcgtctgcgc aaccttcgcc ctcttcctgc agatgctcct ttgccagcac catggctgga
      541 gcgctggaat gacttgactg gacgcttagc acacacagct ggtctgttgg cagcaacagt
      601 ggaggaggaa gcagaggaag ctgctgcagc tttggcagat acaactgctg atcctcggtt
      661 cttggaagct gtcgcatgtt ctagcccacc tgcataccgc gatttgagaa agggtgcacg
      721 gggagcacgc ttaagacgtc aggctgcatc ctacgctcag cgttttgctg ctaagtgtga
      781 aacaatgagc ttttttggtc ctgtaaatta tggtcgtatt gaccctggag ctccagaagc
      841 agcacgctgg acttgggatg gatatgaagc atgtcctgat cgtcgtgcat ttacagcagc
      901 tcgcgtgaca gaccgcatcg aagaagcttt gcttgctgac cctgacgtgg ttgcagctct
      961 ggtaccacgt cgcaaaactg ttttacgccc agcagcacct gatgcaggag cacggcctgc
     1021 aactcgcctg actgctgctg cagatggact gcggactgtc gaacgcctgg gtcacgatct
     1081 tggactggac ccagcagtag cagtcgatgc ttgtcgcaca gcattaactc gtggtctgct
     1141 gactcacgat tggcagccac ctgcaactgt cgctgaccct gtgggttggt tagctcatcg
     1201 cttagatgag agacctggag gtctgtcgcc tcgcttacgt gaactggtgc gtgatatgca
     1261 tcgtatttta gctgactatc ctgctgctga tgcacagact aaggtcgctc tgcagggacg
     1321 cttaacagca ttggcagcag gtgctgtgcc agagcctgag ccaggacgcg ctggtggaac
     1381 aggacgtaca gcagcaggtg gtccaggtcg tgctgatcct gctggtgacc tggctggagg
     1441 tccagcttcc ggagcagacc gtggagctag ccatggaggt ggtcctgcag ctgttacttc
     1501 ccctggtggt ccagcttctg ctgcagtcgc tgcagaggca tcggcagcag aacctcgcgc
     1561 agctgcagtg gttagtcctt cgagtgctgc agaacctcgt gctgcagcag ttgtttcacc
     1621 aagttctcct gcagagccac gggctgctgc agttgctgca ggtagctcag gaacaggtcc
     1681 acggacagct gctgacccag cagcaacatc ccatgcaggt acaggtacag gtactggtac
     1741 tgatgctgat gcaggtgcag gtactgctcc tggtgcacca cgttcgggtg ctgctgcacc
     1801 agctcggcct cgcgacactg gaggtgatgg tactcggact cgcttctata atgaccgtgt
     1861 gattattcac gaggctgcac gtggaacttt ggatctgaca gtaggtggtc ctctggctgc
     1921 tgatttgaca acagctgtag ctcctgtgtt agaccttctg gctcacgatg ctgaactgac
     1981 acgcgcagca gctaaccgcg ctgtggctgc tcgtttggga cctggtagaa ctcctctgtt
     2041 ggcagctatg cgcgcatgtg cagatcttcc ttttattcgt agcgatgctc tgactcgcag
     2101 actggcagaa ctgttagcag atccaggtac agatacaggt gcagcagctt tagatctgac
     2161 tggtcgtttg gtcccacctc cacctagtgg tcctccagtg ctgtgttccg tggacgttat
     2221 gattcaaact gctgacttag ctggatatcg gcctggtgtt actcctctga tcctgggtga
     2281 tatacatgac gcagctttgt tgactccttg ggctctgcag ttccacccac gcgctgcagc
     2341 attacgcgca gagcgggatg ctgctgttct ggatacactg ggtgaccaac gcgctgtgac
     2401 tgttgtcgca cgtagaagta ctggattacc acctttagat tttccaggtc ttgtcttaga
     2461 attaggaggt acttccgatc gtgacgatgc accacggatt ggactggacc agttaactgt
     2521 ggtttcagat ggtgaacgcg cagaattacg cgctgcaggt catgatgcac cattgttatt
     2581 tcataacggt gaactggaca cagcatttca tacagcttta gcattgcctc gcatacgccg
     2641 tccaagatta cctgctttag atcacattcc tcggctgcat tttggaaatg cagtggtggc
     2701 tcggcgtcgt tggacagtac ctgctgctga tattgctgca gtgggacagg cacgtggaga
     2761 cgcagaccgg ctgacagcag cagctgcttt gtgtgcagca cgggctctgc ctgagacatt
     2821 cttcgcatct tccgctaacg agcggaagcc tgtctacgtg gacactagct ccccagcttt
     2881 actggattgt tttcttcgct tggcagatac agctgacgtt ctgacattga gcgaggcagc
     2941 tccagaccgt gcagacagtt ggttgcggca tggaggtcag ccagtcgcag cagaattgcg
     3001 ttgtgtatac ctgcgtcgtg ctgcttaact cgagcaccac caccaccacc actgagatcc
     3061 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     3121 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     3181 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3241 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3301 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3361 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3421 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3481 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3541 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3601 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     3661 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3721 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
     3781 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     3841 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     3901 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     3961 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     4021 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     4081 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     4141 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     4201 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     4261 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     4321 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     4381 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     4441 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     4501 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     4561 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     4621 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4681 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4741 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4801 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     4861 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4921 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4981 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5041 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5101 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5161 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5221 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5281 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5341 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5401 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     5461 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     5521 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     5581 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5641 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5701 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     5761 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     5821 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     5881 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     5941 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     6001 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     6061 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     6121 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     6181 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     6241 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     6301 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     6361 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     6421 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     6481 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     6541 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     6601 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     6661 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     6721 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6781 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     6841 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     6901 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     6961 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     7021 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     7081 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     7141 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     7201 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     7261 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     7321 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     7381 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     7441 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     7501 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     7561 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     7621 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     7681 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     7741 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     7801 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     7861 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     7921 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     7981 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcat
//
