LOCUS       sequence-207271-        6712 bp    DNA     circular SYN 15-APR-2025
DEFINITION  pSKB3 with TEV-cleavable, N-terminal His-tagged SaGGR-I206F-L377H 
            mutant gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6712)
  AUTHORS   Kung Y, McAndrew RP, Xie X, Liu CC, Pereira JH, Adams PD, Keasling 
            JD
  TITLE     Constructing tailored isoprenoid products by structure-guided 
            modification of geranylgeranyl reductase.
  JOURNAL   Structure. 2014 Jul 8;22(7):1028-36. doi: 10.1016/j.str.2014.05.007.
            Epub 2014 Jun 19.
  PUBMED    24954619
REFERENCE   2  (bases 1 to 6712)
  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: 
            "10.1016/j.str.2014.05.007"; journalName: "Structure"; date: 
            "2014-07-8- 8"; volume: "22"; issue: "7"; pages: "1028-36"
COMMENT     Sequence Label: pSKB3-SaGGR-I206F-L377H (JBEI-7202)
FEATURES             Location/Qualifiers
     source          1..6712
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             139..159
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1573..1590
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1643..1661)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1657..1704
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1741..2196
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1828..1847)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2038..2059
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2289..3104)
                     /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(2289..3104)
                     /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     3012..3031
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3096..3115
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3226..3814
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3715..3734
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3968..3985
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4000..4142
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4068..4087)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4228..4250
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4244..4435)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5244..6326)
                     /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     6288..6307
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6327..6404)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6610..6629
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacgat
      121 tacgatatcc caacgaccga aaacctttac ttccagggcc aatggcatat gaaggaactt
      181 aaatatgacg ttctgataat cggaggaggt tttgctggtt cttcagctgc ttaccaatta
      241 tcacgaagag gtcttaaaat attattagta gatagcaaac cgtggaacag gataggagac
      301 aaaccttgtg gagatgcagt aagtaaggct cattttgata agttaggtat gccttatcct
      361 aaaggtgaag agttagaaaa taaaataaat ggaataaagc tttacagccc tgatatgcag
      421 acagtatgga cagtaaatgg agagggtttt gaactaaatg ccccactata taatcaaaga
      481 gttcttaaag aggcacagga tagaggagtc gaaatatggg atttgacaac agcaatgaaa
      541 cccatatttg aagatggtta tgtaaaagga gcagtcctct ttaatagaag aaccaatgag
      601 gaacttacag tatactctaa ggtagttgtt gaggctacag gatattcaag aagtttcaga
      661 agtaaactac cacctgaatt acccataacc gaagatctag atgataagga tgcagacgta
      721 gcgtacagag aggttctact aactaaggaa gatatagagg accatgacta cttgagaata
      781 ttctttgatc aagagacatc tccaggcggt tattggtggt acttccctaa agggaagaac
      841 aaagtcaatg tcggtcttgg aattcagggc ggtatgggat acccaagcat tcatgagtat
      901 tataaaaaat atctagataa gtatgcacct gatgtggata aaagtaaatt attggttaaa
      961 ggaggtgcat tggttcccac cagaagaccg ttatatacaa tggcatggaa cggtataata
     1021 gttataggag attcagggtt tactgtaaat cctgttcatg gcggaggaaa aggttcagcc
     1081 atgatatctg gatattgtgc agcgaaagct atactttcag cttttgagac aggagatttc
     1141 tcagcttcag gattatggga catgaacatt tgctatgtaa atgagtatgg agcaaaacaa
     1201 gccagcctag acatatttag gaggttcctt cagaaactta gtaatgacga cataaactat
     1261 ggaatgaaga agaagattat aaaagaagag gatctgcatg aagcaagtga aaaaggagat
     1321 cttcacttgt ctgttgctga taaggcaatg agagtaatct ctggtttagg cagaccctct
     1381 ttattattca agctaaaggc ggttgcagag tctatgaaga aaataaagga attatatctg
     1441 aattatccaa gatccccctc atctttaggt agttggagaa gagaagttga caatgttcta
     1501 acagagttta acaaaagttt aagttaggat ccgaattcga gctccgtcga caagcttgcg
     1561 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     1621 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     1681 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     1741 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1801 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1861 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1921 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1981 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2041 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2101 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2161 acgcgaattt taacaaaata ttaacgttta caatttcagg tggcactttt cggggaaatg
     2221 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2281 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     2341 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     2401 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     2461 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     2521 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     2581 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     2641 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     2701 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     2761 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     2821 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     2881 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     2941 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     3001 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     3061 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     3121 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     3181 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3241 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3301 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3361 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3421 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3481 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3541 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3601 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3661 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3721 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3781 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3841 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3901 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3961 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     4021 tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac aatctgctct
     4081 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     4141 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4201 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4261 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     4321 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     4381 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     4441 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     4501 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     4561 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     4621 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     4681 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     4741 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     4801 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     4861 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     4921 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     4981 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     5041 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     5101 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     5161 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     5221 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5281 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     5341 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     5401 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     5461 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     5521 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     5581 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     5641 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     5701 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     5761 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5821 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5881 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5941 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     6001 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     6061 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     6121 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     6181 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     6241 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     6301 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     6361 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     6421 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     6481 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     6541 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     6601 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     6661 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa at
//
