LOCUS       sequence-163393-        6789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  stable overexpression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6789)
  AUTHORS   Pacold ME, Brimacombe KR, Chan SH, Rohde JM, Lewis CA, Swier LJ, 
            Possemato R, Chen WW, Sullivan LB, Fiske BP, Cho S, Freinkman E, 
            Birsoy K, Abu-Remaileh M, Shaul YD, Liu CM, Zhou M, Koh MJ, Chung H,
            Davidson SM, Luengo A, Wang AQ, Xu X, Yasgar A, Liu L, Rai G, 
            Westover KD, Vander Heiden MG, Shen M, Gray NS, Boxer MB, Sabatini 
            DM
  TITLE     A PHGDH inhibitor reveals coordination of serine synthesis and 
            one-carbon unit fate.
  JOURNAL   Nat Chem Biol. 2016 Jun;12(6):452-8. doi: 10.1038/nchembio.2070. 
            Epub 2016 Apr 25.
  PUBMED    27110680
REFERENCE   2  (bases 1 to 6789)
  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: 
            "10.1038/nchembio.2070"; journalName: "Nat Chem Biol"; date: 
            "2016-06"; volume: "12"; issue: "6"; pages: "452-8"
COMMENT     Sequence Label: pET30-2-PSAT1
FEATURES             Location/Qualifiers
     source          1..6789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    39..63
                     /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)."
     CDS             112..129
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1301..1319)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1315..1362
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1600..1618)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1686..1790
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1791..1859,1860..2651)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2009..2028)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2822..3410
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3311..3330
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3564..3581
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3596..3735
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3661..3680)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3821..3843
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3837..4028)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5340..6422)
                     /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     6384..6403
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6423..6500)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6706..6725
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
       61 tcccctctag aaataatttt gtttaaactt taagaaggag atatacatat gcatcatcat
      121 catcatcaca tggacgcccc caggcaggtg gtcaactttg ggcctggtcc cgccaagctg
      181 ccgcactcag tgttgttaga gatacaaaag gaattattag actacaaagg agttggcatt
      241 agtgttcttg aaatgagtca caggtcatca gattttgcca agattattaa caatacagag
      301 aatcttgtgc gggaattgct agctgttcca gacaactata aggtgatttt tctgcaagga
      361 ggtgggtgcg gccagttcag tgctgtcccc ttaaacctca ttggcttgaa agcaggaagg
      421 tgtgcggact atgtggtgac aggagcttgg tcagctaagg ccgcagaaga agccaaaaag
      481 tttgggacta taaatatcgt tcaccctaaa cttgggagtt atacaaaaat tccagatcca
      541 agcacctgga acctcaaccc agatgcctcc tacgtgtatt attgcgcaaa tgagacggtg
      601 catggtgtgg agtttgactt tatacccgat gtcaagggag cagtactggt ttgtgacatg
      661 tcctcaaact tcctgtccaa gccagtggat gtttccaagt ttggtgtgat ttttgctggt
      721 gcccagaaga atgttggctc tgctggggtc accgtggtga ttgtccgtga tgacctgctg
      781 gggtttgccc tccgagagtg cccctcggtc ctggaataca aggtgcaggc tggaaacagc
      841 tccttgtaca acacgcctcc atgtttcagc atctacgtca tgggcttggt tctggagtgg
      901 attaaaaaca atggaggtgc cgcggccatg gagaagctta gctccatcaa atctcaaaca
      961 atttatgaga ttattgataa ttctcaagga ttctacgttt gtccagtgga gccccaaaat
     1021 agaagcaaga tgaatattcc attccgcatt ggcaatgcca aaggagatga tgctttagaa
     1081 aaaagatttc ttgataaagc tcttgaactc aatatgttgt ccttgaaagg gcataggtct
     1141 gtgggaggca tccgggcctc tctgtataat gctgtcacaa ttgaagacgt tcagaagctg
     1201 gccgccttca tgaaaaaatt tttggagatg catcagctat gagcggccgc ggatccggct
     1261 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     1321 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     1381 tccggatatc ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat
     1441 ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct
     1501 gactgcgtta gcaatttaac tgtgataaac taccgcatta aagctagctt atcgatgata
     1561 agctgtcaaa catgagaatt aattcttgaa gacgaaaggg cctcgtgata cgcctatttt
     1621 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     1681 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1741 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     1801 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     1861 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     1921 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     1981 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg
     2041 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     2101 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     2161 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     2221 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     2281 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     2341 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     2401 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     2461 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     2521 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     2581 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     2641 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     2701 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     2761 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     2821 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2881 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     2941 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3001 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3061 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3121 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3181 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3241 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3301 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3361 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3421 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3481 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3541 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     3601 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     3661 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     3721 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3781 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3841 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     3901 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     3961 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     4021 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     4081 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     4141 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     4201 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     4261 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     4321 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     4381 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     4441 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag
     4501 gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg agatggcgga cgcgatggat
     4561 atgttctgcc aagggttggt ttgcgcattc acagttctcc gcaagaattg attggctcca
     4621 attcttggag tggtgaatcc gttagcgagg tgccgccggc ttccattcag gtcgaggtgg
     4681 cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg gcggcgccta
     4741 caatccatgc caacccgttc catgtgctcg ccgaggcggc ataaatcgcc gtgacgatca
     4801 gcggtccaat gatcgaagtt aggctggtaa gagccgcgag cgatccttga agctgtccct
     4861 gatggtcgtc atctacctgc ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc
     4921 cggaagcgag aagaatcata atggggaagg ccatccagcc tcgcgtcgcg aacgccagca
     4981 agacgtagcc cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     5041 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     5101 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     5161 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     5221 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     5281 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     5341 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5401 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     5461 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     5521 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     5581 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     5641 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     5701 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     5761 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     5821 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     5881 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     5941 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     6001 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     6061 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     6121 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     6181 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     6241 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     6301 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     6361 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     6421 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     6481 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     6541 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     6601 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     6661 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     6721 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     6781 gaggatcga
//
