LOCUS       sequence-212809-        6708 bp    DNA     circular SYN 15-DEC-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 6708)
  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 6708)
  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: 
            "10.1016/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..6708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     940..959
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(979..1056)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1262..1281
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1365..1384
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1365..1383
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1384..1408
                     /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             1423..1445
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1464..1481
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1503..1523
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2937..2954
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3007..3025)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3021..3068
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3105..3560
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3192..3211)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3402..3423
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3652..4467)
                     /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(3652..4467)
                     /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     4375..4394
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4459..4478
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4589..5177
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5078..5097
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5331..5348
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5363..5502
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5428..5447)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5588..5610
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5604..5795)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6604..6708,1..978))
                     /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"
ORIGIN
        1 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
       61 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
      121 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
      181 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
      241 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
      301 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
      361 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
      421 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
      481 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
      541 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
      601 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
      661 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
      721 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
      781 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
      841 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
      901 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
      961 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     1021 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     1081 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     1141 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     1201 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     1261 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     1321 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     1381 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     1441 ggagatatac catgggcagc agccatcatc atcatcatca cgattacgat atcccaacga
     1501 ccgaaaacct ttacttccag ggccaatggc atatgaagga acttaaatat gacgttctga
     1561 taatcggagg aggttttgct ggttcttcag ctgcttacca attatcacga agaggtctta
     1621 aaatattatt agtagatagc aaaccgtgga acaggatagg agacaaacct tgtggagatg
     1681 cagtaagtaa ggctcatttt gataagttag gtatgcctta tcctaaaggt gaagagttag
     1741 aaaataaaat aaatggaata aagctttaca gccctgatat gcagacagta tggacagtaa
     1801 atggagaggg ttttgaacta aatgccccac tatataatca aagagttctt aaagaggcac
     1861 aggatagagg agtcgaaata tgggatttga caacagcaat gaaacccata tttgaagatg
     1921 gttatgtaaa aggagcagtc ctctttaata gaagaaccaa tgaggaactt acagtatact
     1981 ctaaggtagt tgttgaggct acaggatatt caagaagttt cagaagtaaa ctaccacctg
     2041 aattacccat aaccgaagat ctagatgata aggatgcaga cgtagcgtac agagaggttc
     2101 tactaactaa ggaagatata gaggaccatg actacttgag aatattcttt gatcaagaga
     2161 catctccagg cggttattgg tggtacttcc ctaaagggaa gaacaaagtc aatgtcggtc
     2221 ttggaattca gggcggtatg ggatacccaa gcattcatga gtattataaa aaatatctag
     2281 ataagtatgc acctgatgtg gataaaagta aattattggt taaaggaggt gcattggttc
     2341 ccaccagaag accgttatat acaatggcat ggaacggtat aatagttata ggagattcag
     2401 ggtttactgt aaatcctgtt catggcggag gaaaaggttc agccatgata tctggatatt
     2461 gtgcagcgaa agctatactt tcagcttttg agacaggaga tttctcagct tcaggattat
     2521 gggacatgaa catttgctat gtaaatgagt atggagcaaa acaagccagc ctagacatat
     2581 ttaggaggtt ccttcagaaa cttagtaatg acgacataaa ctatggaatg aagaagaaga
     2641 ttataaaaga agaggatctg catgaagcaa gtgaaaaagg agatcttcac ttgtctgttg
     2701 ctgataaggc aatgagagta atctctggtt taggcagacc ctctttatta ttcaagctaa
     2761 aggcggttgc agagtctatg aagaaaataa aggaattata tctgaattat ccaagatccc
     2821 cctcatcttt aggtagttgg agaagagaag ttgacaatgt tctaacagag tttaacaaaa
     2881 gtttaagtta ggatccgaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc
     2941 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     3001 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     3061 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     3121 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3181 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3241 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3301 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3361 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3421 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3481 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3541 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3601 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     3661 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     3721 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     3781 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     3841 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     3901 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     3961 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     4021 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     4081 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     4141 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     4201 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     4261 catctcatct gtaacatcat tggcaacgct acctttgcca tgcttcagaa acaactctgg
     4321 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     4381 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     4441 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     4501 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4561 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4621 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4681 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     4741 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4801 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4861 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4921 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4981 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5041 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5101 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5161 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5221 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5281 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     5341 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     5401 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5461 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     5521 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5581 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     5641 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     5701 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     5761 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     5821 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     5881 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     5941 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     6001 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     6061 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     6121 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     6181 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     6241 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     6301 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     6361 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     6421 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     6481 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     6541 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     6601 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     6661 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttt
//
