LOCUS       Exported                6499 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Bacterial expression for structure determination; may not be full 
            ORF.
ACCESSION   .
VERSION     .
KEYWORDS    MAPK3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6499)
  TITLE     SGC Oxford
REFERENCE   2  (bases 1 to 6499)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6499
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     regulatory      153..162
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1360..1377
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1430..1448)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1444..1491
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1528..1983
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1615..1634)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1825..1846
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2076..2891)
                     /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     2799..2818
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2883..2902
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3013..3601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3502..3521
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3755..3772
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3787..3929
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3855..3874)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4015..4037
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4031..4222)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5031..6113)
                     /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     6075..6094
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6114..6191)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6397..6416
                     /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 atccatggcg gcggcggcgg ctcagggggg
      181 cgggggcggg gagccccgta gaaccgaggg ggtcggcccg ggggtcccgg gggaggtgga
      241 gatggtgaag gggcagccgt tcgacgtggg cccgcgctac acgcagttgc agtacatcgg
      301 cgagggcgcg tacggcatgg tcagctcggc ctatgaccac gtgcgcaaga ctcgcgtggc
      361 catcaagaag atcagcccct tcgaacatca gacctactgc cagcgcacgc tccgggagat
      421 ccagatcctg ctgcgcttcc gccatgagaa tgtcatcggc atccgagaca ttctgcgggc
      481 gtccaccctg gaagccatga gagatgtcta cattgtgcag gacctgatgg agactgacct
      541 gtacaagttg ctgaaaagcc agcagctgag caatgaccat atctgctact tcctctacca
      601 gatcctgcgg ggcctcaagt acatccactc cgccaacgtg ctccaccgag atctaaagcc
      661 ctccaacctg ctcatcaaca ccacctgcga ccttaagatt tgtgatttcg gcctggcccg
      721 gattgccgat cctgagcatg accacaccgg cttcctgacg gagtatgtgg ctacgcgctg
      781 gtaccgggcc ccagagatca tgctgaactc caagggctat accaagtcca tcgacatctg
      841 gtctgtgggc tgcattctgg ctgagatgct ctctaaccgg cccatcttcc ctggcaagca
      901 ctacctggat cagctcaacc acattctggg catcctgggc tccccatccc aggaggacct
      961 gaattgtatc atcaacatga aggcccgaaa ctacctacag tctctgccct ccaagaccaa
     1021 ggtggcttgg gccaagcttt tccccaagtc agactccaaa gcccttgacc tgctggaccg
     1081 gatgttaacc tttaacccca ataaacggat cacagtggag gaagcgctgg ctcaccccta
     1141 cctggagcag tactatgacc cgacggatga gccagtggcc gaggagccct tcaccttcgc
     1201 catggagctg gatgacctac ctaaggagcg gctgaaggag ctcatcttcc aggagacagc
     1261 acgcttccag cccggagtgc tggaggcccc ctagtgacag taaaggtgga tacggatccg
     1321 aattcgagct ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga
     1381 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     1441 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     1501 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     1561 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1621 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1681 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1741 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1801 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1861 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1921 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     1981 tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2041 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     2101 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     2161 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     2221 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     2281 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     2341 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     2401 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     2461 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     2521 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     2581 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     2641 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     2701 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     2761 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     2821 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     2881 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     2941 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3001 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3061 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3121 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3181 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3241 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3301 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3361 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3421 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3481 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3541 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3601 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3661 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3721 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3781 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatata
     3841 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     3901 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     3961 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4021 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     4081 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     4141 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     4201 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     4261 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     4321 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     4381 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     4441 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     4501 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     4561 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     4621 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     4681 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     4741 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     4801 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     4861 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     4921 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     4981 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     5041 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5101 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     5161 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     5221 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     5281 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     5341 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     5401 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     5461 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     5521 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     5581 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     5641 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     5701 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     5761 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     5821 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     5881 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     5941 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     6001 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     6061 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     6121 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     6181 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     6241 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     6301 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     6361 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     6421 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     6481 gatctcgatc ccgcgaaat
//
