LOCUS       sequence-305619-        8103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-AmmB4 in E. Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8103)
  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 8103)
  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-ammB4
FEATURES             Location/Qualifiers
     source          1..8103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     46..65
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     149..168
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        149..167
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    168..192
                     /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             207..229
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             248..265
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             275..292
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3116..3133
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3186..3204)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3200..3247
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3284..3739
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3371..3390)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3581..3602
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3831..4646)
                     /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(3831..4646)
                     /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     4554..4573
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4638..4657
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4768..5356
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5257..5276
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5510..5527
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5542..5681
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5607..5626)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5767..5789
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5783..5974)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6783..7865)
                     /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     7827..7846
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7866..7943)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
       61 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      121 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      181 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      241 cagcagccat catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatgat
      301 gacttccaca cgcacagcgc atccaactga tccgggtcgt gccgatgcac cagcacccgg
      361 tgtagacggg gccggcagcc cagtacctga cccagatggc gcacgtggat ctgcgcccgg
      421 tgcggcagct gcagatgaag gagccgcgcc agcacctggt gcggatggcg ccggctcgcc
      481 tgtccctgac tcaggcggcg ttggtggctc ggcaccccgt gcggccgctg cggatgaagg
      541 agccgcaccc gctccagggg cgggccgcgc ctcgggcgtg gcggcagcgg cagaagtacc
      601 ttctcctcaa tcaggtcgcc cacctggtgg tcgtggtaca gcgccgcgtc cgcgtcatgc
      661 ggctgacccg ggcactacag cgccgttggt ttggtcggtc cctcatggac cgggtaccct
      721 gactattcgt cttcttacac ctggcctttt acgtactccg ggttttccag cgcatttact
      781 tgagccagtg acggctccgg caacgaccgc gctggcagca gaggctgatg gtcgtgctac
      841 ccgcgaagaa gcagatcgtc gtgcgtttct cgatgatcgt tggccggctt tacgcgcgac
      901 agcgcgcgca gatggggcgc gtcatccggc ctggcgcgcc ttattacgcg ctcaccgcct
      961 cgttaccgaa gggcgtctcc ttgatcccgg ggcggaagcg gccttgacag cagtgggtgg
     1021 accggcactc actgcatggg caggcgaatg gaatgcacgt cgcgcagatg atgcagcgtt
     1081 tcgcgatcgt gccgcggctg cagtgcgtgc ggcaacggca gatgcgtatc gtgcgactga
     1141 tcgtgcagta gatgatcccc gtgttcgcca tgcggttttc gtaagcaatc cggattttct
     1201 ggatacagca ttaaccccta cgccccttgc acgctatggt acggggccag atggtgttcc
     1261 cccacgtcgt gcacgtcgcg cgctggcaac ggctcatcgt taccttcgtc gtcttacgac
     1321 tcgttgtgaa actgtgagct tctttggtcc ggtgttattt acgggttttg atagcggacg
     1381 cccggaacca gtattacgcg gcgcccctgc agaagaacgt gttttagtag aagcgtcgac
     1441 gtggctcgtt gaagcattgg gtgaagattt gacggcgcgt actccaccag gcttgcgtat
     1501 tgcgcgtcgt caccccttat ttcgtccttt agatgatgga cgcggtcttg agcgcgtggt
     1561 ggatggcaaa cgttttcgtt tggatccggc ggcgttggcc ctgtggtgtg cagcggatgg
     1621 tggaacggcg ttggatgccc tggctgctgg cttagggttg gatccggctg cgcgtgatcg
     1681 tgcagtacgt gcgctgggcc caacccttct tttaacgggt cacccgttgc cggcaacctg
     1741 cctggatccg acacatcgtt tggcggcgca tgatccagcc ggacctgccg ctgcattggc
     1801 agaagcgctt gcagcgcatg cgcgcacgcc atggccagag cgtactgcgt cgtatggtac
     1861 ggtgcgtgct atggctcgtg acttggccgg tggtgaacgt aaagcagctg gagcgcatta
     1921 tgcggatcgc gaagcattgt ttgaagatcg cgtatcacct catagtgaac gcctcacatt
     1981 tggcggtcct gttgtagatg gaattcgccg tgcgctttct gcggttcttc cattatgtcg
     2041 tttggctgcg ttggcggcgc gcgaagatgc gcgtgatacg gtacgtcgcg ccacaggtgg
     2101 agtagcggtg ccttttgtac gtcttgctcg cgcagagtta ccgccagata ctccgcgttt
     2161 agatcgtctt cgcgcggtgc tccgtgatct tgttgcagcc ggcaccccag atgcggatgg
     2221 tgctgtccat ttaacggcgg cggctgtgga agaagcaaca gcggatctct ggcatgatat
     2281 tcctgccgcg gcacgcgcgg gagcctcttt gccttcgccg gatcttatgg ctgtagggcc
     2341 agatttagcg cgtgctactt ggctcttatc tgagcttcat gatgattgtt catcggtcta
     2401 tggcggtctt gaacgtcgtg cccattcgga tcctgatggt ctctgggatg gatttctccg
     2461 cacggttagt gcacatttac caccaggcgg tatggcgacg gtagtgagtc gccgccgttc
     2521 tgcgcacgtg actccagagc ttccaggttt atcggtagaa ctttctggac tctctgcaaa
     2581 tgatcgtgca gaaacattgc cgattgccga agtagaagtt gcggccgatg gttcagcgct
     2641 tttagctcgt ggtggacgtc gcttgttgta tcccggcgat ctccatagcc ccctgcatcg
     2701 cgccgtgtct cttcctgcat tggttcctgt tgatgttgaa actggtgatc gtacaccccg
     2761 tcttgtcatt gatggcgttg tttatcaacg tgcacgttgg cgcgtacctc ttccgcccgc
     2821 ggctggcggt gaaccttatg atcgttggct ggcggttcaa cgttggcgtc tgggccatgg
     2881 actcccccgc cgtgtatttc tccgtcatcc ggaagaacct aagccgctgt atgtagattt
     2941 tgcagatcct ttagcagttg cagaaaccgc ggcactgccg cctgccacgg tgcttgtggg
     3001 tgaaatgtta ccggccccag gtgagttgtg gtggtcgacg ggcgatgggg ctcaatgtgc
     3061 ggaatttcgc ctggggtgtg ttgtttggcc agccgaagaa ggacgctaac tcgagcacca
     3121 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     3181 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     3241 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     3301 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3361 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3421 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3481 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3541 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3601 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3661 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3721 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3781 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     3841 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     3901 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     3961 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     4021 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     4081 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     4141 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     4201 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     4261 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     4321 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     4381 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     4441 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     4501 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     4561 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     4621 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     4681 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4741 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4801 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4861 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     4921 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4981 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5041 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5101 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5161 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5221 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5281 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5341 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5401 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5461 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     5521 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     5581 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     5641 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     5701 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     5761 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     5821 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     5881 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     5941 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     6001 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     6061 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     6121 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     6181 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     6241 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     6301 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     6361 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     6421 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     6481 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     6541 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     6601 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     6661 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     6721 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     6781 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     6841 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     6901 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     6961 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     7021 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     7081 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     7141 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     7201 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     7261 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     7321 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     7381 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     7441 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     7501 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     7561 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     7621 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     7681 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     7741 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     7801 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     7861 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     7921 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     7981 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     8041 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     8101 gaa
//
