LOCUS       sequence-141328-        6491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of PSAT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6491)
  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 6491)
  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-PSAT
FEATURES             Location/Qualifiers
     source          1..6491
                     /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(1365..1382)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1392..1409)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1428..1450
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1465..1489
                     /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(1489..1508)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1490..1508)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1592..1611)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1817..1894
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1895..2977
                     /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(1914..1933)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3786..3977
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3971..3993)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4079..4221
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4134..4153
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4236..4253)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4407..4995)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4487..4506)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5106..5125)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5117..5932
                     /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             5117..5932
                     /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(5190..5209)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6025..6480)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6162..6183)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6374..6393
                     /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 tccactagta acggccgcca gtgtgctgga attcgccctt
      241 ttggatcctc atagctgatg catctccaaa aattttttca tgaaggcggc cagcttctga
      301 acgtcttcaa ttgtgacagc attatacaga gaggcccgga tgcctcccac agacctatgc
      361 cctttcaagg acaacatatt gagttcaaga gctttatcaa gaaatctttt ttctaaagca
      421 tcatctcctt tggcattgcc aatgcggaat ggaatattca tcttgcttct attttggggc
      481 tccactggac aaacgtagaa tccttgagaa ttatcaataa tctcataaat tgtttgagat
      541 ttgatggagc taagcttctc catggccgcg gcacctccat tgtttttaat ccactccaga
      601 accaagccca tgacgtagat gctgaagcat ggaggcgtgt tgtacaagga gctgtttcca
      661 gcctgcacct tgtattccag gaccgagggg cactctcgga gggcaaaccc cagcaggtca
      721 tcacggacaa tcaccacggt gaccccagca gagccaacat tcttctgggc accagcaaaa
      781 atcacaccaa acttggaaac atccactggc ttggacagga agtttgagga catgtcacaa
      841 accagtactg ctcccttgac atcgggtata aagtcaaact ccacaccatg caccgtctca
      901 tttgcgcaat aatacacgta ggaggcatct gggttgaggt tccaggtgct tggatctgga
      961 atttttgtat aactcccaag tttagggtga acgatattta tagtcccaaa cttcttggct
     1021 tcttctgcgg ccttagctga ccaagctcct gtcaccacat agtccgcaca ccttcctgct
     1081 ttcaagccaa tgaggtttaa ggggacagca ctgaactggc cgcacccacc tccttgcaga
     1141 aaaatcacct tatagttgtc tggaacagct agcaattccc gcacaagatt ctctgtattg
     1201 ttaataatct tggcaaaatc tgatgacctg tgactcattt caagaacact aatgccaact
     1261 cctttgtagt ctaataattc cttttgtatc tctaacaaca ctgagtgcgg cagcttggcg
     1321 ggaccaggcc caaagttgac cacctgcctg ggggcgtcca tatggctgcc gcgcggcacc
     1381 aggccgctgc tgtgatgatg atgatgatgg ctgctgccca tggtatatct ccttcttaaa
     1441 gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag
     1501 tcgtattaat ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc
     1561 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
     1621 gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc
     1681 cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg
     1741 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
     1801 gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
     1861 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     1921 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     1981 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     2041 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     2101 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     2161 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     2221 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     2281 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     2341 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     2401 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     2461 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     2521 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     2581 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     2641 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     2701 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     2761 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     2821 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     2881 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     2941 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     3001 agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca
     3061 gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
     3121 tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct
     3181 ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc
     3241 tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta
     3301 tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc
     3361 taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt
     3421 gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt
     3481 tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga
     3541 tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc
     3601 gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt
     3661 taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca
     3721 tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg
     3781 catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga
     3841 cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg
     3901 aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg
     3961 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     4021 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     4081 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
     4141 agagcagatt gtactgagag tgcaccatat atgcggtgtg aaataccgca cagatgcgta
     4201 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4261 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4321 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4381 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4441 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4501 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4561 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4621 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4681 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4741 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4801 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     4861 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4921 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4981 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5041 gaaaactcac gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag
     5101 taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt
     5161 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     5221 atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa
     5281 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     5341 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     5401 gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga
     5461 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     5521 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     5581 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     5641 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
     5701 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     5761 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     5821 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     5881 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat
     5941 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6001 catttccccg aaaagtgcca cctgaaattg taaacgttaa tattttgtta aaattcgcgt
     6061 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     6121 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     6181 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     6241 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     6301 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     6361 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     6421 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     6481 cccattcgcc a
//
