LOCUS       sequence-130610-        6358 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6358)
  AUTHORS   Hari SB, Merritt EA, Maly DJ
  TITLE     Sequence determinants of a specific inactive protein kinase 
            conformation.
  JOURNAL   Chem Biol. 2013 Jun 20;20(6):806-15. doi: 
            10.1016/j.chembiol.2013.05.005.
  PUBMED    23790491
REFERENCE   2  (bases 1 to 6358)
  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.chembiol.2013.05"; journalName: "Chem Biol"; date: 
            "2013-06-20- 20"; volume: "20"; issue: "6"; pages: "806-15"
COMMENT     Sequence Label: Erk2
FEATURES             Location/Qualifiers
     source          1..6358
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..37)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     178..200
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(194..385)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1194..2276)
                     /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     2238..2257
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2277..2354)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2560..2579
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2667..2686
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2667..2685
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2686..2710
                     /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             2725..2747
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2758..2775
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2800..2820
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3859..3894
                     /codon_start=1
                     /product="epitope tag derived from MAP kinase 2"
                     /label=Tag-100
                     /translation="EETARFQPGYRS"
     CDS             3961..3978
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4031..4049)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4045..4092
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4216..4235)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4402..4505
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4506..4574,4575..5366)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4724..4743)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5537..6125
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6026..6045
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6279..6296
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(6311..6358,1..92)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
       61 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
      121 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      181 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
      241 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
      301 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
      361 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
      421 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
      481 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
      541 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
      601 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
      661 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
      721 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
      781 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
      841 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
      901 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
      961 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     1021 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     1081 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     1141 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     1201 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     1261 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     1321 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     1381 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     1441 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     1501 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     1561 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     1621 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     1681 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     1741 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     1801 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     1861 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     1921 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     1981 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     2041 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     2101 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     2161 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     2221 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     2281 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     2341 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     2401 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     2461 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     2521 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     2581 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     2641 cgagatcgat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     2701 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgcac
     2761 catcatcatc atcattcttc tggtgtagat ctgggtaccg agaacctgta cttccaatcc
     2821 aatgcggcgg cggcggcggc gggcccggag atggtccgcg ggcaggtgtt cgacgtgggg
     2881 ccgcgctaca ctaatctctc gtacatcgga gaaggcgcct acggcatggt ttgttctgct
     2941 tatgataatc tcaacaaagt tcgagttgct atcaagaaaa tcagtccttt tgagcaccag
     3001 acctactgtc agagaaccct gagagagata aaaatcctac tgcgcttcag acatgagaac
     3061 atcatcggca tcaatgacat catccgggca ccaaccattg agcagatgaa agatgtatat
     3121 atagtacagg acctcatgga gacagatctt tacaagctct tgaagacaca gcacctcagc
     3181 aatgatcata tctgctattt tctttatcag atcctgagag gattaaagta tatacattca
     3241 gctaatgttc tgcaccgtga cctcaagcct tccaacctcc tgctgaacac cacttgtgat
     3301 ctcaagatct gtgactttgg ccttgcccgt gttgcagatc cagaccatga tcatacaggg
     3361 ttcttgacag agtatgtagc cacgcgttgg tacagagctc cagaaattat gttgaattcc
     3421 aagggttata ccaagtccat tgatatttgg tctgtgggct gcatcctggc agagatgcta
     3481 tccaacaggc ctatcttccc aggaaagcat taccttgacc agctgaatca catcctgggt
     3541 attcttggat ctccatcaca ggaagatctg aattgtataa taaatttaaa agctagaaac
     3601 tatttgcttt ctctcccgca caaaaataag gtgccgtgga acagattgtt cccaaacgct
     3661 gactccaaag ctctggattt actggataaa atgttgacat ttaaccctca caagaggatt
     3721 gaagttgaac aggctctggc ccacccgtac ctggagcagt attatgaccc aagtgatgag
     3781 cccattgctg aagcaccatt caagtttgac atggagctgg acgacttacc taaggagaag
     3841 ctcaaagaac tcatttttga agagactgct cgattccagc caggatacag atcttaacat
     3901 tggaagtgga taacggatcc gaattcgagc tccgtcgaca agcttgcggc cgcactcgag
     3961 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     4021 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     4081 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     4141 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4201 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4261 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4321 acctcgaccc caaaaaactt gattagggtg atggttcaca ttaacgctta caatttaggt
     4381 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     4441 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataatt tgaaaaagga
     4501 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4561 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4621 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4681 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4741 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4801 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4861 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4921 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4981 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5041 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5101 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5161 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     5221 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     5281 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5341 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     5401 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5461 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5521 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5581 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5641 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     5701 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5761 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5821 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5881 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5941 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     6001 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     6061 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     6121 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     6181 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     6241 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     6301 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccg
//
