LOCUS       sequence-137188-        6448 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination; may not be full 
            ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6448)
  TITLE     SGC Oxford
REFERENCE   2  (bases 1 to 6448)
  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"
COMMENT     Sequence Label: MAPK8
FEATURES             Location/Qualifiers
     source          1..6448
                     /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             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1309..1326
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1379..1397)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1393..1440
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1477..1932
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1564..1583)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1774..1795
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2025..2840)
                     /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(2025..2840)
                     /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     2748..2767
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2832..2851
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2962..3550
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3451..3470
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3704..3721
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3736..3878
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3804..3823)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3964..3986
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3980..4171)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4980..6062)
                     /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     6024..6043
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6063..6140)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6346..6365
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatgagc agaagcaagc gtgacaacaa
      181 tttttatagt gtagagattg gagattctac attcacagtc ctgaaacgat atcagaattt
      241 aaaacctata ggctcaggag ctcaaggaat agtatgcgca gcttatgatg ccattcttga
      301 aagaaatgtt gcaatcaaga agctaagccg accatttcag aatcagactc atgccaagcg
      361 ggcctacaga gagctagttc ttatgaaatg tgttaatcac aaaaatataa ttggcctttt
      421 gaatgttttc acaccacaga aatccctaga agaatttcaa gatgtttaca tagtcatgga
      481 gctcatggat gcaaatcttt gccaagtgat tcagatggag ctagatcatg aaagaatgtc
      541 ctaccttctc tatcagatgc tgtgtggaat caagcacctt cattctgctg gaattattca
      601 tcgggactta aagcccagta atatagtagt aaaatctgat tgcactttga agattcttga
      661 cttcggtctg gccaggactg caggaacgag ttttatgatg acgccttatg tagtgactcg
      721 ctactacaga gcacccgagg tcatccttgg catgggctac aaggaaaacg tggatttatg
      781 gtctgtgggg tgcattatgg gagaaatggt ttgccacaaa atcctctttc caggaaggga
      841 ctatattgat cagtggaata aagttattga acagcttgga acaccatgtc ctgaattcat
      901 gaagaaactg caaccaacag taaggactta cgttgaaaac agacctaaat atgctggata
      961 tagctttgag aaactcttcc ctgatgtcct tttcccagct gactcagaac acaacaaact
     1021 taaagccagt caggcaaggg atttgttatc caaaatgctg gtaatagatg catctaaaag
     1081 gatctctgta gatgaagctc tccaacaccc gtacatcaat gtctggtatg atccttctga
     1141 agcagaagct ccaccaccaa agatccctga caagcagtta gatgaaaggg aacacacaat
     1201 agaagagtgg aaagaattga tatataagga agttatggac ttgtgacagt aaaggtggat
     1261 acggatccga attcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac
     1321 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1381 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1441 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     1501 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1561 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1621 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1681 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1741 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1801 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1861 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1921 cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1981 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     2041 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     2101 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     2161 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     2221 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     2281 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     2341 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     2401 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     2461 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     2521 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     2581 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     2641 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     2701 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     2761 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     2821 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     2881 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2941 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3001 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3061 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3121 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3181 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3241 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3301 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3361 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3421 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3481 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3541 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3601 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3661 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3721 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3781 ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     3841 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     3901 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3961 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4021 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     4081 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     4141 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     4201 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     4261 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     4321 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     4381 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     4441 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     4501 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     4561 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     4621 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     4681 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     4741 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     4801 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     4861 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     4921 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     4981 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5041 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     5101 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     5161 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     5221 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     5281 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     5341 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     5401 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     5461 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     5521 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     5581 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     5641 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     5701 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     5761 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     5821 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     5881 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     5941 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     6001 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     6061 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     6121 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     6181 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     6241 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     6301 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     6361 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     6421 gaggatcgag atctcgatcc cgcgaaat
//
