LOCUS       Exported                6885 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pET-28a-hPKM2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6885)
  AUTHORS   Christofk HR, Vander Heiden MG, Wu N, Asara JM, Cantley LC
  TITLE     Pyruvate kinase M2 is a phosphotyrosine-binding protein.
  JOURNAL   Nature. 2008 Mar 13;452(7184):181-6. doi: 10.1038/nature06667.
  PUBMED    18337815
REFERENCE   2  (bases 1 to 6885)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6885
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     36..55
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        36..54
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    55..79
                     /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             94..116
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             135..152
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             162..179
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1785..1802
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1855..1873)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1869..1916
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1953..2408
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2040..2059)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2250..2271
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2500..3315)
                     /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     3223..3242
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3307..3326
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3437..4025
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3926..3945
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4179..4196
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4211..4350
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4276..4295)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4436..4458
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4452..4643)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5452..6534)
                     /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     6496..6515
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6535..6612)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6818..6837
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt
       61 gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata
      121 ccatgggcag cagccatcat catcatcatc acagcagcgg cctggtgccg cgcggcagcc
      181 atatgtctaa accgcactct gaagctggta ccgccttcat tcagacccag caactgcacg
      241 cagccatggc tgacacattc ctggagcaca tgtgccgcct ggacattgat tcaccaccca
      301 tcacagcccg gaacactggc atcatctgta ccattggccc agcttcccga tcagtggaga
      361 cgttgaagga gatgattaag tctggaatga atgtggctcg tctgaacttc tctcatggaa
      421 ctcatgagta ccatgcggag accatcaaga atgtgcgcac agccacggaa agctttgctt
      481 ctgaccccat cctctaccgg cccgttgctg tggctctaga cactaaagga cctgagatcc
      541 gaactgggct catcaagggc agcggcactg cagaggtgga gctgaagaag ggagccactc
      601 tcaaaatcac gctggataac gcctacatgg aaaagtgtga cgagaacatc ctgtggctgg
      661 actacaagaa catctgcaag gtggtggaag tgggcagcaa gatctacgtg gatgatgggc
      721 ttatttctct ccaggtgaag cagaaaggtg ccgacttcct ggtgacggag gtggaaaatg
      781 gtggctcctt gggcagcaag aagggtgtga accttcctgg ggctgctgtg gacttgcctg
      841 ctgtgtcgga gaaggacatc caggatctga agtttggggt cgagcaggat gttgatatgg
      901 tgtttgcgtc attcatccgc aaggcatctg atgtccatga agttaggaag gtcctgggag
      961 agaagggaaa gaacatcaag attatcagca aaatcgagaa tcatgagggg gttcggaggt
     1021 ttgatgaaat cctggaggcc agtgatggga tcatggtggc tcgtggtgat ctaggcattg
     1081 agattcctgc agagaaggtc ttccttgctc agaagatgat gattggacgg tgcaaccgag
     1141 ctgggaagcc tgtcatctgt gctactcaga tgctggagag catgatcaag aagccccgcc
     1201 ccactcgggc tgaaggcagt gatgtggcca atgcagtcct ggatggagcc gactgcatca
     1261 tgctgtctgg agaaacagcc aaaggggact atcctctgga ggctgtgcgc atgcagcacc
     1321 tgattgcccg tgaggcagag gctgccatct accacttgca attatttgag gaactccgcc
     1381 gcctggcgcc cattaccagc gaccccacag aagccaccgc cgtgggtgcc gtggaggcct
     1441 ccttcaagtg ctgcagtggg gccataatcg tcctcaccaa gtctggcagg tctgctcacc
     1501 aggtggccag ataccgccca cgtgccccca tcattgctgt gacccggaat ccccagacag
     1561 ctcgtcaggc ccacctgtac cgtggcatct tccctgtgct gtgcaaggac ccagtccagg
     1621 aggcctgggc tgaggacgtg gacctccggg tgaactttgc catgaatgtt ggcaaggccc
     1681 gaggcttctt caagaaggga gatgtggtca ttgtgctgac cggatggcgc cctggctccg
     1741 gcttcaccaa caccatgcgt gttgttcctg tgccgtgact cgagcaccac caccaccacc
     1801 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     1861 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     1921 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     1981 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2041 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2101 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2161 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2221 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2281 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2341 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     2401 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2461 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     2521 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     2581 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     2641 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     2701 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     2761 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     2821 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     2881 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     2941 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     3001 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     3061 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     3121 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     3181 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     3241 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     3301 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     3361 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3421 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3481 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3541 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3601 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3661 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3721 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3781 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3841 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3901 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3961 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4021 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4081 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4141 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4201 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4261 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     4321 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     4381 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4441 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     4501 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     4561 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     4621 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     4681 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     4741 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     4801 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     4861 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     4921 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     4981 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     5041 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     5101 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     5161 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     5221 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     5281 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     5341 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     5401 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     5461 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5521 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     5581 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     5641 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     5701 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     5761 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     5821 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     5881 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     5941 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     6001 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     6061 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     6121 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     6181 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     6241 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     6301 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     6361 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     6421 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     6481 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     6541 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     6601 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     6661 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     6721 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     6781 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     6841 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtc
//
