LOCUS       sequence-141318-        6977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of N-terminal His-tagged.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6977)
  AUTHORS   Mullarky E, Lucki NC, Beheshti Zavareh R, Anglin JL, Gomes AP, 
            Nicolay BN, Wong JC, Christen S, Takahashi H, Singh PK, Blenis J, 
            Warren JD, Fendt SM, Asara JM, DeNicola GM, Lyssiotis CA, Lairson 
            LL, Cantley LC
  TITLE     Identification of a small molecule inhibitor of 3-phosphoglycerate 
            dehydrogenase to target serine biosynthesis in cancers.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Feb 16;113(7):1778-83. doi: 
            10.1073/pnas.1521548113. Epub 2016 Feb 1.
  PUBMED    26831078
REFERENCE   2  (bases 1 to 6977)
  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: 
            "10.1073/pnas.1521548113"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2016-02-16- 16"; volume: "113"; issue: "7"; pages: "1778-83"
COMMENT     Sequence Label: pET28a-PHGDH
FEATURES             Location/Qualifiers
     source          1..6977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(207..239)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             complement(1851..1868)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1878..1895)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1914..1936
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1951..1975
                     /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)."
     primer_bind     complement(1975..1994)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1976..1994)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2078..2097)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2303..2380
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2381..3463
                     /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     complement(2400..2419)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4272..4463
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4457..4479)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4565..4707
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4620..4639
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4722..4739)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4893..5481)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4973..4992)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5592..5611)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5603..6418
                     /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             5603..6418
                     /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     complement(5676..5695)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6511..6966)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6648..6669)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6860..6879
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccgcgaccc atttgctgtc caccagtcat gctagccata
      241 tgttagaagt ggaactggaa ggcttcagtc acatgctgct tccacgcttc caggctgggc
      301 agcaaggagg agatgcccat gacgtgccag gtctccccat ctgacaccag tgaagtctgg
      361 taggacagca gccgcacgcc tgcctctgcc aggaggccaa tcatggtagg cagcattgca
      421 gggtcagagg tctgagtccg gaataggagc aggggcaggt ccctgcggag aggcacttct
      481 ggcctgaaga cagctccatt gagcccctgc agtacaggtg tagtgccttg gaccaagccc
      541 acagcctggt aaggggcgcc tgccagggcc acggccagga ggcattcccc gaagccttgc
      601 tcccctggtg cagcagggct gtgggaggtg gtgacattga ggccagcctc tttcaccagc
      661 agcttagcgt tcaccaagtt cacatccgcc tgcttggaag cctctttcag gaggccgaca
      721 atgactgcgg ggcttaggca gttcccagca ttcttcaggg atgttccctg tgttatcacc
      781 tggatggtcc ctttggggga cccagcccag gctcgcatca gtgtccccag agcttctgcc
      841 agaccaatcc aaggcttggt gtgtggagag aaggcactgg taagggcctg ggcattcaca
      901 acccccgtga gagatttccc cttcaccatg tccacgaact gaacagcaat ttcctcccca
      961 cagcggctct gagcctcctt ggtgctggca cccaggtggg gacagctgat gacattctca
     1021 tggtccacca aggcccggtc ccgtggcggc tcttccgtaa acacgtccag tgcagccccg
     1081 gcacactggc cagactgcag ggcccggagc agggcgcctt cgtccacgat ccctccacgg
     1141 gcacagttca ccacacgcac ccccttcttg cactgggcaa aggtgttgtc attcagcaag
     1201 cctgtcgtgg agggcaggag aggagtgtgc acagtgatga aatcacagag aggccagatc
     1261 tcctccaggg gcagctgctg aacaccaaag gaggccgaga cctctgggga aatgatgggg
     1321 tcatacccta tagtcttcat cccaaaggac tgcatccggg tagctacctc tctcccaatc
     1381 ctgcccaggc caagaattcc cagggtcttt ccattcagct ctgttcccat gaacttcttc
     1441 cgctcccatt tgccgtcctt catcgaagcc gtcgcctggg gaatctgcct ggccaggcac
     1501 atgatcattc cacaagtgag ttctgcggca ctgaggctgt tcccattggg ggtgttcata
     1561 accaagatgc ccttccttgt tgcggcctcc agatccacat tgtccacacc tgtgccagcc
     1621 ctgcccacca cctggagttt ctcagctgcg ttgatgacat cagcggtcac cttggtggca
     1681 gagcgaacaa taaggccttc acagtcctgc agctccgcta tcagctcctc tttgctaagg
     1741 ttctgctttt ccaccacctg cagccctcca tcttgcaaga tcttccggca gcaagggtcc
     1801 aggctgtcac tgatgagcac tttccgcaga tttgcaaaag ccatcatatg gctgccgcgc
     1861 ggcaccaggc cgctgctgtg atgatgatga tgatggctgc tgcccatggt atatctcctt
     1921 cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat
     1981 agtgagtcgt attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg
     2041 gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat
     2101 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
     2161 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
     2221 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
     2281 aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc
     2341 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     2401 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     2461 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     2521 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     2581 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     2641 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     2701 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     2761 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     2821 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     2881 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     2941 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     3001 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     3061 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     3121 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     3181 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     3241 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     3301 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     3361 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     3421 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     3481 taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc
     3541 cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct
     3601 tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg
     3661 accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc
     3721 acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg
     3781 ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga
     3841 cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc
     3901 gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct
     3961 tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt
     4021 tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa
     4081 cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca
     4141 gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg
     4201 tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca
     4261 cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa
     4321 gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca
     4381 tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg
     4441 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     4501 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     4561 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     4621 ggcatcagag cagattgtac tgagagtgca ccatatatgc ggtgtgaaat accgcacaga
     4681 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     4741 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4801 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4861 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4921 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4981 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5041 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5101 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5161 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5221 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5281 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     5341 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5401 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5461 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5521 tggaacgaaa actcacgtta agggattttg gtcatgaaca ataaaactgt ctgcttacat
     5581 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctctaggcc
     5641 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     5701 cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt
     5761 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     5821 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     5881 tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata
     5941 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     6001 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     6061 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     6121 gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt
     6181 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     6241 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     6301 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     6361 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga
     6421 attaattcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6481 cgcgcacatt tccccgaaaa gtgccacctg aaattgtaaa cgttaatatt ttgttaaaat
     6541 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     6601 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     6661 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     6721 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     6781 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     6841 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     6901 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     6961 gcgcgtccca ttcgcca
//
