LOCUS       sequence-114714-        7978 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a cysteine-free version of pET30Xa-SBL1(160E) (4Cys to 
            Ser) with N-term Histag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7978)
  AUTHORS   Coffa G, Imber AN, Maguire BC, Laxmikanthan G, Schneider C, Gaffney 
            BJ, Brash AR
  TITLE     On the relationships of substrate orientation, hydrogen abstraction,
            and product stereochemistry in single and double dioxygenations by 
            soybean lipoxygenase-1 and its Ala542Gly mutant.
  JOURNAL   J Biol Chem. 2005 Nov 18;280(46):38756-66. Epub 2005 Sep 12.
  PUBMED    16157595
REFERENCE   2  (bases 1 to 7978)
  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"; journalName: "J Biol 
            Chem."; date: "2005-11-18"; volume: "280(46)"; pages: "38756-66. 
            Epub 2005 Sep 12"
COMMENT     Sequence Label: pET30Xa-SBL1(160E,NoCys)
FEATURES             Location/Qualifiers
     source          1..7978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1990..1999
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2610..2627
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2680..2698)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2694..2741
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2778..3233
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2865..2884)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3075..3096
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3325..4140)
                     /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(3325..4140)
                     /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     4048..4067
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4132..4151
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4262..4850
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4751..4770
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5004..5021
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5036..5175
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5101..5120)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5261..5283
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5277..5468)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6277..7359)
                     /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     7321..7340
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7360..7437)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7643..7662
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7750..7769
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7750..7768
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7769..7793
                     /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             7808..7830
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7841..7858
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7868..7885
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             7892..7936
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             7967..7978
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 atgttttcag caggccataa gatcaaaggg acagtggtgt tgatgcccaa gaatgagttg
       61 gaagttaacc ctgatggctc agcagttgac aaccttaatg ctttcttggg ccgtagtgtc
      121 tcccttcagc tcattagtgc taccaaggct gatgcacatg gaaaaggaaa agttggaaag
      181 gatacgttct tggaagggat taatacttcg ttaccaactt tgggagcagg agagtctgca
      241 ttcaatattc attttgaatg ggacggaagc atgggaatcc ccggtgcgtt ttacataaag
      301 aactacatgc aagttgagtt cttcctcaag agtttgactc ttgaagccat ttcaaaccaa
      361 ggaaccatcc gctttgtttc caactcatgg gtttataaca ctaaacttta caaaagcgtg
      421 cgcattttct ttgccaacca tacatatgta cctagtgaga caccagcacc acttgtggag
      481 tacagagaag aagaattgaa gagtttaaga ggaaatggaa cgggagagcg caaggaatat
      541 gataggatct atgattatga tgtctacaat gatttgggca acccagataa gagtgaaaag
      601 ttagctcgtc cagttcttgg agggtctagc acctttccct accctcgtag gggaagaact
      661 ggtcgaggtc caacagttac agacccgaat actgagaaac aaggcgaagt attttatgtt
      721 ccaagagatg aaaatttagg tcacttgaag tctaaggatg ctcttgagat tggaacaaaa
      781 tctttatctc aaattgtcca gccggcgttc gaatctgcgt tcgatttgaa atccacgcca
      841 attgagtttc atagcttcca agatgtgcat gacttgtatg aaggtggaat taagcttcct
      901 agagatgtaa ttagcacaat tatcccctta ccagtgatca aagaactcta tcgtaccgat
      961 ggtcaacaca tcctcaagtt tccacaacct cacgtcgttc aagtgagtca gtctgcatgg
     1021 atgactgatg aagaatttgc tagagagatg attgctggtg taaatccctc cgtaattcgt
     1081 ggtcttgaag agttccctcc aaaaagtaat ctggatcctg caatctatgg tgatcaaagc
     1141 agtaagataa cagcagattc ccttgatcta gatgggtaca caatggacga ggcacttggt
     1201 agtcgaaggt tatttatgtt agattaccat gatatcttca tgccatatgt gaggcagata
     1261 aatcagctga attctgccaa gacttatgcg acaaggacta tccttttttt gagagaagat
     1321 ggaactttaa agccagtggc catcgaatta agtttgccac attctgctgg ggatctgtca
     1381 gctgccgtca gtcaagtcgt cttacctgct aaggaaggtg ttgagagcac aatttggcta
     1441 ctagccaaag cttatgtcat cgtaaatgac tcttcctacc atcaactcat gagccattgg
     1501 ttaaatactc atgcggcgat ggagccattc gtcatagcaa cacaccgaca tcttagcgtg
     1561 cttcacccaa tttacaagct tctgactcct cactatcgta acaacatgaa catcaacgca
     1621 cttgccaggc aatctctaat taatgctaat ggcataatag agacaacctt tttgccctca
     1681 aagtattctg tggagatgtc ttcggcggtt tataagaatt gggttttcac tgatcaagca
     1741 ctacctgcag atcttatcaa gagaggagtg gcaattaagg atccatcaac cccacatgga
     1801 gttcgtcttc tgatagagga ctatccttat gccgctgatg gactggagat atgggctgca
     1861 attaagacat gggttcaaga atatgtgccc ttgtactatg caagagatga tgatgtcaag
     1921 aatgattctg aactccaaca ttggtggaaa gaagctgtag agaaaggcca tggtgatttg
     1981 aaagacaagc catggtggcc taagttgcag acacttgaag accttgttga agtttccctc
     2041 attatcatat ggattgcttc agctctccat gcagccgtta attttggtca gtatccttat
     2101 ggaggtttga taatgaaccg cccaactgct tctagaaggt tgcttcctga gaaaggcacc
     2161 ccagaatatg aagaaatgat taataaccat gaaaaggctt atttgagaac aattacatca
     2221 aagttgccga ctctcattag cctttcagtg atagagatct tgtcgacaca tgcttctgat
     2281 gaggtctacc ttggccagag ggacaaccca cattggacct ctgattcaaa agcattacaa
     2341 gcctttcaaa agtttggaaa caagctcaag gaaattgagg aaaaacttgt gaggaggaac
     2401 aatgatccga gtctgcaggg caatcgactt ggcccggttc aactgccata cactttgctt
     2461 tatcctagca gtgaggaagg gttaactttt aggggaattc caaatagcat ctctatctaa
     2521 actagtcatg gggctctaac tctcctctgg ccatggcgat atcggatccg aattcgagct
     2581 ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg
     2641 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     2701 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     2761 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2821 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2881 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2941 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3001 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3061 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3121 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3181 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     3241 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3301 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     3361 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     3421 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     3481 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     3541 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     3601 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     3661 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     3721 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     3781 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     3841 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     3901 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     3961 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     4021 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     4081 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     4141 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     4201 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4261 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4321 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4381 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4441 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4501 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4561 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4621 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4681 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4741 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4801 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4861 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4921 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4981 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     5041 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     5101 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     5161 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5221 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5281 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     5341 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     5401 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     5461 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     5521 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     5581 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     5641 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     5701 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     5761 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     5821 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     5881 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     5941 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     6001 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     6061 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     6121 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     6181 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     6241 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     6301 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6361 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     6421 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     6481 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     6541 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     6601 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     6661 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     6721 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     6781 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     6841 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     6901 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     6961 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     7021 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     7081 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     7141 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     7201 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     7261 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     7321 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     7381 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     7441 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     7501 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     7561 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     7621 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     7681 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc
     7741 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     7801 aaataatttt gtttaacttt aagaaggaga tatacatatg caccatcatc atcatcattc
     7861 ttctggtctg gtgccacgcg gttctggtat gaaagaaacc gctgctgcta aattcgaacg
     7921 ccagcacatg gacagcccag atctgggtac cggtggtggc tccggtattg agggtcgc
//
