LOCUS       sequence-305612-        7791 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-AmmB3 in E. Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7791)
  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 7791)
  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-ammB3
FEATURES             Location/Qualifiers
     source          1..7791
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     32..51
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     135..154
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        135..153
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    154..178
                     /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             193..215
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             234..251
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             261..278
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2790..2807
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2860..2878)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2874..2921
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2958..3413
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3045..3064)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3255..3276
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3505..4320)
                     /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(3505..4320)
                     /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     4228..4247
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4312..4331
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4442..5030
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4931..4950
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5184..5201
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5216..5355
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5281..5300)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5441..5463
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5457..5648)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6457..7539)
                     /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     7501..7520
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7540..7617)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
       61 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
      121 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
      181 tctagaaata attttgttta actttaagaa ggagatatac catgggcagc agccatcatc
      241 atcatcatca cagcagcggc ctggtgccgc gcggcagcca tatgatgaca cgtgtttgga
      301 ccttggggac agtgtttgtt gtgcgccatg ccggtatgcc gtttgattgg ttagaaagtc
      361 ttggtggtag cgcggatctc catgaggctg cagagcgttt attggatgct gaagaagaac
      421 tccgcgcggc agcgggcagt gcgtttccgc gcttggcggc ggcggtggaa gaatgtgctc
      481 ctgaacgttt gcccggtgtg ccagcaccgg cacgtgaagc ggctcgtgcg tggcgtgcgg
      541 cggcggcccg ttatcgtgaa gctcatcgta cggccgatga agcagcgacg aaagagttgc
      601 gccgtttgtt agaacgtccg gaagtcgcgg aagcggtctt gttaagcaat ccggatgcgt
      661 tacgcaatat gttacgtcct ttactggcgc atgatgggcc cctcacgtca cgctatcgtc
      721 gcgcgcgtcg ccaactttat acgtatgttc aacgcttttg tgcgaagaat gaaacagttt
      781 ccttctttgg cccgatggct tatggagaag tggacccagg gcttccgact gcggatggtg
      841 cactcttgag tactcgcctt ccgcgtacac gccgtgtatt tgtgagccat tgggcaggtc
      901 gtgcgctctt gcgcgctgtt acgcgtgata cagctttgtt accggattta ccgtttcatc
      961 gtacgcgtga tggtgagccg gcgtggcctg atggagccag cccggaagat gcggccgcgg
     1021 cacgtacagt tttgaccgca ctgacgccgg atggtacgac tcttcgtggt ctggtgcgta
     1081 ttttaggtac gccggcacgt gaactcgctc gtgccctgcg tctcttggtg gcagcagatg
     1141 ctgctgatgt gggtcttggt ggtggaccgt ttgatctgga gcctctgacg actttgcgtg
     1201 atggtctgga tcgccttccc ccttcggcag cacgtgatcg ctggattgct cgtattgaag
     1261 gtcttgaagc gttgcgcgcc gctctgcaag ctgctggacc ggatgagcgc gaagcggcta
     1321 ttggcgcgtt agaacgcgcg tttacggaag cgacgggtga gccggcacgt cgtgcagcgg
     1381 ggtcgacgta tgctgatcgt gcagtgtttt atgaagaagg tagctcgccg tttgcactcc
     1441 gcattggtcc tgccttagcg gcgcgttggg aacaacgctt acgtggtgtg ttggaactgt
     1501 gtaccgcgca tggtcatctt actcaacgcg cggcggcaga tgcggtccgt agcgcgctgg
     1561 gtacgggcgc gcgcttagat ttggaacgct atgccgcggc ggctgcagaa gcttttccgg
     1621 cgccgggttc tgcatttgca gtggcacatg ccccggatta tcctgccgcc gatcgtgagc
     1681 gccatacaac tcgtctccgt gaattagcgg atggtcttcg tggtgatcgt tatgcattgg
     1741 tggatttgtg tcctcgcgct tctggcgcgg cgggtctggc tgatgcagat ctggtggtgg
     1801 cgcgtgttca tcatcactta cttgtgccgg gttggctgga tacgatgcat ccggagccgc
     1861 gtcgttttgc tgccgcagca gcagattggg tagcgcgtca agatggacgc gtggttggtt
     1921 ttgattttgg tcgtcgtaat aaaggttatt atcgctttcc tggtcgtgca gtagctctgc
     1981 gtccggcgtc atggactgat gcgggtgatc cacctcttcg tccgcgtgat ctgactgtgg
     2041 aacttggtga accccctggt cctgccgcag gctcgggtca tggttatgaa acgggtccgg
     2101 gttatgaacc gggctatggc cgcggcagcg caagcggatc aggtactggc gtacgccttc
     2161 tcgacccaga tggtcgtgaa gtgacggcat atattccgtt gaatgatttt gttaaatatg
     2221 caccatttgc cgctctctca catcctcaag tactgcatcc cgcctttaca gcgccggaag
     2281 ctgcgccaga ggcagcgccg caagtagcct ctgatgcagc gcgtgatgca gctccagatg
     2341 atccacctgg tggtgcgcct ggtaccgcac atggtggtac cgccccggta gatgctgcgc
     2401 atggtggaac cgatccaggg gcacttccag aagtggctgt tgatggtgtg gttttacaac
     2461 gtccgcgttg gcgtgttgat gcgagccgtt tacgtcgcag cgctcctcat gcacgtttct
     2521 tagagttacg tcgtttggcg cgtcgtacgg gtttacgttt tctgttttgt cgtgcaccgc
     2581 gtgaacgtaa accctatctg ttggatttag cctcaccgtt agcggctgat cttgcggctc
     2641 acattggaca tcgtgaagat gtactcacgg ctgaagccat gtcaccagcc ccgggtgatt
     2701 tgtggttgcg tgatgaacaa ggtcgtcgtt atacatgcga attgcgtatg caatttacgg
     2761 gtcgtgaagt ggcggcgccc taactcgagc accaccacca ccaccactga gatccggctg
     2821 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     2881 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     2941 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3001 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3061 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3121 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3181 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3241 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3301 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3361 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     3421 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3481 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     3541 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     3601 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     3661 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     3721 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     3781 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     3841 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     3901 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     3961 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     4021 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     4081 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     4141 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     4201 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     4261 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     4321 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     4381 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4441 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4501 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4561 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4621 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4681 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4741 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4801 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4861 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4921 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4981 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5041 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5101 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5161 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     5221 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     5281 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     5341 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5401 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5461 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     5521 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     5581 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     5641 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     5701 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     5761 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     5821 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     5881 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     5941 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     6001 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     6061 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     6121 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     6181 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     6241 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     6301 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     6361 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     6421 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     6481 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6541 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     6601 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     6661 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     6721 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     6781 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     6841 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     6901 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     6961 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     7021 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     7081 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     7141 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     7201 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     7261 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     7321 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     7381 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     7441 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     7501 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     7561 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     7621 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     7681 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     7741 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg a
//
