LOCUS       sequence-212817-        6708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pSKB3 with TEV-cleavable, N-terminal His-tagged SaGGR-F219L 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-F219L (JBEI-7186)
FEATURES             Location/Qualifiers
     source          1..6708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     956..975
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(995..1072)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1278..1297
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1381..1400
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1381..1399
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1400..1424
                     /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             1439..1461
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1480..1497
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1519..1539
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2953..2970
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3023..3041)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3037..3084
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3121..3576
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3208..3227)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3418..3439
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3668..4483)
                     /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(3668..4483)
                     /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     4391..4410
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4475..4494
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4605..5193
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5094..5113
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5347..5364
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5379..5518
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5444..5463)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5604..5626
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5620..5811)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6620..6708,1..994))
                     /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 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
       61 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
      121 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
      181 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
      241 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
      301 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
      361 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
      421 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
      481 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
      541 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
      601 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
      661 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
      721 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
      781 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
      841 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
      901 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
      961 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     1021 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     1081 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     1141 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     1201 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     1261 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     1321 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     1381 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     1441 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacgat
     1501 tacgatatcc caacgaccga aaacctttac ttccagggcc aatggcatat gaaggaactt
     1561 aaatatgacg ttctgataat cggaggaggt tttgctggtt cttcagctgc ttaccaatta
     1621 tcacgaagag gtcttaaaat attattagta gatagcaaac cgtggaacag gataggagac
     1681 aaaccttgtg gagatgcagt aagtaaggct cattttgata agttaggtat gccttatcct
     1741 aaaggtgaag agttagaaaa taaaataaat ggaataaagc tttacagccc tgatatgcag
     1801 acagtatgga cagtaaatgg agagggtttt gaactaaatg ccccactata taatcaaaga
     1861 gttcttaaag aggcacagga tagaggagtc gaaatatggg atttgacaac agcaatgaaa
     1921 cccatatttg aagatggtta tgtaaaagga gcagtcctct ttaatagaag aaccaatgag
     1981 gaacttacag tatactctaa ggtagttgtt gaggctacag gatattcaag aagtttcaga
     2041 agtaaactac cacctgaatt acccataacc gaagatctag atgataagga tgcagacgta
     2101 gcgtacagag aggttctact aactaaggaa gatatagagg accatgacta cttgagaata
     2161 ttcatagatc aagagacatc tccaggcggt tattggtggt acctgcctaa agggaagaac
     2221 aaagtcaatg tcggtcttgg aattcagggc ggtatgggat acccaagcat tcatgagtat
     2281 tataaaaaat atctagataa gtatgcacct gatgtggata aaagtaaatt attggttaaa
     2341 ggaggtgcat tggttcccac cagaagaccg ttatatacaa tggcatggaa cggtataata
     2401 gttataggag attcagggtt tactgtaaat cctgttcatg gcggaggaaa aggttcagcc
     2461 atgatatctg gatattgtgc agcgaaagct atactttcag cttttgagac aggagatttc
     2521 tcagcttcag gattatggga catgaacatt tgctatgtaa atgagtatgg agcaaaacaa
     2581 gccagcctag acatatttag gaggttcctt cagaaactta gtaatgacga cataaactat
     2641 ggaatgaaga agaagattat aaaagaagag gatctgctgg aagcaagtga aaaaggagat
     2701 cttcacttgt ctgttgctga taaggcaatg agagtaatct ctggtttagg cagaccctct
     2761 ttattattca agctaaaggc ggttgcagag tctatgaaga aaataaagga attatatctg
     2821 aattatccaa gatccccctc atctttaggt agttggagaa gagaagttga caatgttcta
     2881 acagagttta acaaaagttt aagttaggat ccgaattcga gctccgtcga caagcttgcg
     2941 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     3001 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     3061 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     3121 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3181 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3241 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3301 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3361 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3421 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3481 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3541 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     3601 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     3661 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     3721 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     3781 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     3841 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     3901 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     3961 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     4021 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     4081 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     4141 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     4201 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     4261 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     4321 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     4381 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     4441 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     4501 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     4561 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4621 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4681 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4741 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4801 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4861 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4921 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4981 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5041 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5101 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5161 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5221 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     5281 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     5341 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     5401 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     5461 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     5521 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5581 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5641 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     5701 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     5761 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     5821 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     5881 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     5941 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     6001 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     6061 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     6121 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     6181 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     6241 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     6301 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     6361 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     6421 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     6481 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     6541 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     6601 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     6661 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgg
//
