LOCUS       sequence-305597-        7713 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-AmmB1 in E. Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7713)
  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 7713)
  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-ammB1
FEATURES             Location/Qualifiers
     source          1..7713
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     124..143
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        124..142
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    143..167
                     /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             182..204
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             223..240
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             250..267
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2701..2718
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2771..2789)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2785..2832
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2869..3324
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2956..2975)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3166..3187
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3416..4231)
                     /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(3416..4231)
                     /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     4139..4158
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4223..4242
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4353..4941
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4842..4861
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5095..5112
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5127..5266
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5192..5211)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5352..5374
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5368..5559)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6368..7450)
                     /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     7412..7431
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7451..7528)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
       61 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
      121 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
      181 ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac
      241 agcagcggcc tggtgccgcg cggcagccat atgatggctg ccacctcagg agaaggtcat
      301 tggcgtttat atcctcgttt attattgcgt cgtgcgggat ttgatattgg gttactgact
      361 ggtctgacgg atcctgaagt ggtaggagca gcagcagggt atcgtgaacg cgctgctgct
      421 tttgagcgtc aacgtgaaca atgtttagcg ggcttacgtg cagaagtgga tgctgctgcg
      481 cgtgatgggg atcgctcgtt attacgctct ttatctaaag cacgtagtcg tattggtcgt
      541 cgtggaccct tagatggccg cttgagcact gttaatgatg ccgctacggc ttatgaacgc
      601 gctcgtcgcg agcgtgaaga tgcgtgggaa gttttgcgtg atgctctgga tgcggaaagt
      661 gcagatcgcc cacgtcgtat tcgtgcggtg ctggatgatg atcgcgtacg tgatgcagta
      721 ctgcaactgg ctccaagcta tgccgctgaa gttgaacgtt ggagcgcagg cccacgtgat
      781 cgtcctgcta atgcgaaaac tcgcgcgttc tttcgtcgca catatctcta ttgtcaacgt
      841 ctgggtgcga agaatgaaac tacgtcgttc tttggaccat taattcatgg aaccgtggcg
      901 gccgatggtg aaggaattac attgggaccg gaaactccta gcgggattac agatacggaa
      961 gcttttctgg cgttttgggc ggtaggggag ttagcgcgtg tcatggcgga agagccggct
     1021 acggcagcgg gccttccgtt gacatggatt gcggcatgtc gtcgtgaaga tggggcatta
     1081 gtgctgccag atggtcgtcg cgtgggcgca cgcggagcct taggtcgcgt tgctgcactg
     1141 gtggatggga ctcgtacgcc gcgtgatatt gctcgtgatt ctggtcttga attagctgaa
     1201 gttacggccg ccgtagaacg tctgcgtcgc gcaggcgccg tgcatcgtca tccagagccc
     1261 ccgtcgactg agcctcgtcc gttggattgg ttaattgcac tggcggatga acgtgcggct
     1321 actacggctt ggcctgaagc gttacgtgat ttgcgtgatc gtgcaggttt gtatgcaggt
     1381 gcggttggtc ccgtaaaacg tcgtcgtgcg cttgaagatg ttgaagcggc ttttgcagcg
     1441 ctgactgggt cggatccccg ccgtgaaggt ggtcgcatgt atgcggatcg ccttgtcgta
     1501 tcacttgata gccaagggga tcaagggcca gtgacggttg gggcagctac ggcggcacgt
     1561 tgggaacgtg agctggcgcc ggtgctggat gtggcggctc attatggaga gctgctgcaa
     1621 tctgccgcat ctcgtttatg tgctgatttg atgcgtgaag cgggtacccg cacgatgcct
     1681 tatgatgaat taattcgccg ttcccgtgct gccgtacaac aaggacgtga tcgtgatcat
     1741 acgggtccgg cagatgcatt tgcagcggag ttaacgcgta cggttactga tgcggtacgt
     1801 cgtcctcatg gggcacccgc gcgcgcagtt ttagcgccgg cagatcttgc ggaacttcgc
     1861 agcccgcgtg cccgtcctcg ctttgttagt cctgatctga tgttggaaac gcgtgaaggg
     1921 cgccctgaaa cactcgtatt aggtgagtta catccatatg tgtttgcgtg gggcagccaa
     1981 ggtttatttg atcatgatcc ggccagcagt cgcgcggcat tttcaggacg ccttgatccg
     2041 tggggcggtc cagaacgtat tgcgacggtg attcgtcgtc gtcgccataa aggattagtg
     2101 gcacattggt ttccggggcg ttttgtggaa attactgcac gtgcgactga tgatcgtgcg
     2161 cgtactttac ccgtgacgga tctgacggtg gagctggctg atggggtgcc ggttttgcgt
     2221 gcaccagatg gcgagctggt gttatatgca ggtgaagatg atcatgtgca ccttcgtgca
     2281 tttgcggcac ctactgcggt tttgccccct gttgtagtgg atggggtgat gccgcgttgg
     2341 ggtgttggcg atgtggttgc acaacgtgca cgttggtggg ttgatcccgc gcgccttttg
     2401 ggtgatggtc ccccgtccgc gacaggggct tatcgtactg tccaacactt gcgtacggag
     2461 ctggcgttac cgcgttttgt gtttgcacac ttagccggtg aaccgaagcc ggttggtgtt
     2521 gatctggatt ccccactggc agtggaaacg ttgactgcct tagtgtcagc gtctgcggcg
     2581 gatcgtgtag cactgacgga aatgcgccct gcaccagatc gcctgtggct ccgtcgtcaa
     2641 gggcgtccgg tgacgagcga atttcgtttg gcattacgtt gggaaactgt ctaactcgag
     2701 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     2761 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     2821 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     2881 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2941 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3001 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3061 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3121 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3181 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3241 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     3301 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     3361 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     3421 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     3481 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     3541 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     3601 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     3661 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     3721 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     3781 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     3841 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     3901 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     3961 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     4021 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     4081 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     4141 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     4201 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     4261 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4321 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4381 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4441 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     4501 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4561 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4621 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4681 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4741 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4801 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4861 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4921 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4981 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     5041 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     5101 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     5161 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5221 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     5281 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5341 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5401 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     5461 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     5521 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     5581 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     5641 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     5701 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     5761 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     5821 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     5881 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     5941 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     6001 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     6061 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     6121 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     6181 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     6241 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     6301 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     6361 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     6421 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     6481 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     6541 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     6601 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     6661 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     6721 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     6781 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     6841 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     6901 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     6961 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     7021 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     7081 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     7141 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     7201 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     7261 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     7321 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     7381 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     7441 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     7501 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     7561 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     7621 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     7681 acgccgaaac aagcgctcat gagcccgaag tgg
//
