LOCUS       sequence-212807-        6708 bp    DNA     circular SYN 15-APR-2025
DEFINITION  SaGGR site-directed mutation of L377H.
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 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: pSKB3SaGGR L377H (JBEI-3683)
FEATURES             Location/Qualifiers
     source          1..6708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     932..951
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(971..1048)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1254..1273
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1357..1376
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1357..1375
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1376..1400
                     /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             1415..1437
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1456..1473
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1495..1515
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2929..2946
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2999..3017)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3013..3060
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3097..3552
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3184..3203)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3394..3415
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3644..4459)
                     /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(3644..4459)
                     /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     4367..4386
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4451..4470
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4581..5169
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5070..5089
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5323..5340
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5355..5494
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5420..5439)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5580..5602
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5596..5787)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6596..6708,1..970))
                     /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 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
       61 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
      121 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
      181 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
      241 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
      301 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
      361 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
      421 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
      481 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
      541 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
      601 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
      661 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
      721 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
      781 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
      841 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
      901 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
      961 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     1021 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     1081 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     1141 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     1201 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     1261 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     1321 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     1381 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     1441 accatgggca gcagccatca tcatcatcat cacgattacg atatcccaac gaccgaaaac
     1501 ctttacttcc agggccaatg gcatatgaag gaacttaaat atgacgttct gataatcgga
     1561 ggaggttttg ctggttcttc agctgcttac caattatcac gaagaggtct taaaatatta
     1621 ttagtagata gcaaaccgtg gaacaggata ggagacaaac cttgtggaga tgcagtaagt
     1681 aaggctcatt ttgataagtt aggtatgcct tatcctaaag gtgaagagtt agaaaataaa
     1741 ataaatggaa taaagcttta cagccctgat atgcagacag tatggacagt aaatggagag
     1801 ggttttgaac taaatgcccc actatataat caaagagttc ttaaagaggc acaggataga
     1861 ggagtcgaaa tatgggattt gacaacagca atgaaaccca tatttgaaga tggttatgta
     1921 aaaggagcag tcctctttaa tagaagaacc aatgaggaac ttacagtata ctctaaggta
     1981 gttgttgagg ctacaggata ttcaagaagt ttcagaagta aactaccacc tgaattaccc
     2041 ataaccgaag atctagatga taaggatgca gacgtagcgt acagagaggt tctactaact
     2101 aaggaagata tagaggacca tgactacttg agaatattca tagatcaaga gacatctcca
     2161 ggcggttatt ggtggtactt ccctaaaggg aagaacaaag tcaatgtcgg tcttggaatt
     2221 cagggcggta tgggataccc aagcattcat gagtattata aaaaatatct agataagtat
     2281 gcacctgatg tggataaaag taaattattg gttaaaggag gtgcattggt tcccaccaga
     2341 agaccgttat atacaatggc atggaacggt ataatagtta taggagattc agggtttact
     2401 gtaaatcctg ttcatggcgg aggaaaaggt tcagccatga tatctggata ttgtgcagcg
     2461 aaagctatac tttcagcttt tgagacagga gatttctcag cttcaggatt atgggacatg
     2521 aacatttgct atgtaaatga gtatggagca aaacaagcca gcctagacat atttaggagg
     2581 ttccttcaga aacttagtaa tgacgacata aactatggaa tgaagaagaa gattataaaa
     2641 gaagaggatc tgcatgaagc aagtgaaaaa ggagatcttc acttgtctgt tgctgataag
     2701 gcaatgagag taatctctgg tttaggcaga ccctctttat tattcaagct aaaggcggtt
     2761 gcagagtcta tgaagaaaat aaaggaatta tatctgaatt atccaagatc cccctcatct
     2821 ttaggtagtt ggagaagaga agttgacaat gttctaacag agtttaacaa aagtttaagt
     2881 taggatccga attcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac
     2941 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     3001 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     3061 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     3121 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3181 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3241 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3301 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3361 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3421 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3481 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3541 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3601 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     3661 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     3721 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     3781 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     3841 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     3901 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     3961 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     4021 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     4081 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     4141 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     4201 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     4261 ctgtaacatc attggcaacg ctacctttgc catgcttcag aaacaactct ggcgcatcgg
     4321 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     4381 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     4441 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     4501 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4561 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4621 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4681 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4741 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4801 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4861 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4921 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4981 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     5041 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     5101 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     5161 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     5221 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     5281 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     5341 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     5401 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     5461 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     5521 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5581 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     5641 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     5701 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5761 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5821 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5881 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5941 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     6001 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     6061 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     6121 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     6181 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     6241 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     6301 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     6361 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     6421 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     6481 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     6541 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     6601 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6661 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttca
//
