LOCUS       sequence-258934-        7713 bp    DNA     circular SYN 15-APR-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 Apr 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"
     CDS             2428..2445
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2498..2516)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2512..2559
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2596..3051
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2683..2702)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2893..2914
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3143..3958)
                     /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(3143..3958)
                     /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     3866..3885
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3950..3969
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4080..4668
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4569..4588
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4822..4839
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4854..4993
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4919..4938)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5079..5101
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5095..5286)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6095..7177)
                     /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     7139..7158
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7178..7255)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7461..7480
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7564..7583
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7564..7582
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7583..7607
                     /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             7622..7644
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7663..7680
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7690..7707
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 atggctgcca cctcaggaga aggtcattgg cgtttatatc ctcgtttatt attgcgtcgt
       61 gcgggatttg atattgggtt actgactggt ctgacggatc ctgaagtggt aggagcagca
      121 gcagggtatc gtgaacgcgc tgctgctttt gagcgtcaac gtgaacaatg tttagcgggc
      181 ttacgtgcag aagtggatgc tgctgcgcgt gatggggatc gctcgttatt acgctcttta
      241 tctaaagcac gtagtcgtat tggtcgtcgt ggacccttag atggccgctt gagcactgtt
      301 aatgatgccg ctacggctta tgaacgcgct cgtcgcgagc gtgaagatgc gtgggaagtt
      361 ttgcgtgatg ctctggatgc ggaaagtgca gatcgcccac gtcgtattcg tgcggtgctg
      421 gatgatgatc gcgtacgtga tgcagtactg caactggctc caagctatgc cgctgaagtt
      481 gaacgttgga gcgcaggccc acgtgatcgt cctgctaatg cgaaaactcg cgcgttcttt
      541 cgtcgcacat atctctattg tcaacgtctg ggtgcgaaga atgaaactac gtcgttcttt
      601 ggaccattaa ttcatggaac cgtggcggcc gatggtgaag gaattacatt gggaccggaa
      661 actcctagcg ggattacaga tacggaagct tttctggcgt tttgggcggt aggggagtta
      721 gcgcgtgtca tggcggaaga gccggctacg gcagcgggcc ttccgttgac atggattgcg
      781 gcatgtcgtc gtgaagatgg ggcattagtg ctgccagatg gtcgtcgcgt gggcgcacgc
      841 ggagccttag gtcgcgttgc tgcactggtg gatgggactc gtacgccgcg tgatattgct
      901 cgtgattctg gtcttgaatt agctgaagtt acggccgccg tagaacgtct gcgtcgcgca
      961 ggcgccgtgc atcgtcatcc agagcccccg tcgactgagc ctcgtccgtt ggattggtta
     1021 attgcactgg cggatgaacg tgcggctact acggcttggc ctgaagcgtt acgtgatttg
     1081 cgtgatcgtg caggtttgta tgcaggtgcg gttggtcccg taaaacgtcg tcgtgcgctt
     1141 gaagatgttg aagcggcttt tgcagcgctg actgggtcgg atccccgccg tgaaggtggt
     1201 cgcatgtatg cggatcgcct tgtcgtatca cttgatagcc aaggggatca agggccagtg
     1261 acggttgggg cagctacggc ggcacgttgg gaacgtgagc tggcgccggt gctggatgtg
     1321 gcggctcatt atggagagct gctgcaatct gccgcatctc gtttatgtgc tgatttgatg
     1381 cgtgaagcgg gtacccgcac gatgccttat gatgaattaa ttcgccgttc ccgtgctgcc
     1441 gtacaacaag gacgtgatcg tgatcatacg ggtccggcag atgcatttgc agcggagtta
     1501 acgcgtacgg ttactgatgc ggtacgtcgt cctcatgggg cacccgcgcg cgcagtttta
     1561 gcgccggcag atcttgcgga acttcgcagc ccgcgtgccc gtcctcgctt tgttagtcct
     1621 gatctgatgt tggaaacgcg tgaagggcgc cctgaaacac tcgtattagg tgagttacat
     1681 ccatatgtgt ttgcgtgggg cagccaaggt ttatttgatc atgatccggc cagcagtcgc
     1741 gcggcatttt caggacgcct tgatccgtgg ggcggtccag aacgtattgc gacggtgatt
     1801 cgtcgtcgtc gccataaagg attagtggca cattggtttc cggggcgttt tgtggaaatt
     1861 actgcacgtg cgactgatga tcgtgcgcgt actttacccg tgacggatct gacggtggag
     1921 ctggctgatg gggtgccggt tttgcgtgca ccagatggcg agctggtgtt atatgcaggt
     1981 gaagatgatc atgtgcacct tcgtgcattt gcggcaccta ctgcggtttt gccccctgtt
     2041 gtagtggatg gggtgatgcc gcgttggggt gttggcgatg tggttgcaca acgtgcacgt
     2101 tggtgggttg atcccgcgcg ccttttgggt gatggtcccc cgtccgcgac aggggcttat
     2161 cgtactgtcc aacacttgcg tacggagctg gcgttaccgc gttttgtgtt tgcacactta
     2221 gccggtgaac cgaagccggt tggtgttgat ctggattccc cactggcagt ggaaacgttg
     2281 actgccttag tgtcagcgtc tgcggcggat cgtgtagcac tgacggaaat gcgccctgca
     2341 ccagatcgcc tgtggctccg tcgtcaaggg cgtccggtga cgagcgaatt tcgtttggca
     2401 ttacgttggg aaactgtcta actcgagcac caccaccacc accactgaga tccggctgct
     2461 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     2521 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     2581 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2641 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2701 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2761 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2821 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2881 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2941 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3001 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     3061 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3121 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     3181 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     3241 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     3301 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     3361 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     3421 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     3481 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     3541 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3601 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     3661 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3721 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3781 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     3841 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3901 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     3961 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     4021 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4081 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4141 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4201 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4261 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4321 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4381 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4441 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4501 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4561 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4621 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4681 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4741 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4801 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4861 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     4921 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4981 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     5041 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5101 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     5161 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     5221 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     5281 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     5341 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     5401 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     5461 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     5521 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     5581 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     5641 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     5701 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     5761 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     5821 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     5881 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     5941 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     6001 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     6061 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     6121 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     6181 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     6241 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     6301 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     6361 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     6421 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     6481 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     6541 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     6601 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     6661 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     6721 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     6781 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     6841 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     6901 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     6961 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     7021 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     7081 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     7141 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     7201 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     7261 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     7321 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     7381 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     7441 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     7501 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     7561 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     7621 ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac
     7681 agcagcggcc tggtgccgcg cggcagccat atg
//
