LOCUS       sequence-163398-        6361 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 6361)
  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 6361)
  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-PSPH
FEATURES             Location/Qualifiers
     source          1..6361
                     /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(873..891)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      887..934
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1172..1190)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1258..1362
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1363..1431,1432..2223)
                     /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(1581..1600)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2394..2982
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2883..2902
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3136..3153
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3168..3307
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3233..3252)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3393..3415
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3409..3600)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4912..5994)
                     /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     5956..5975
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5995..6072)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6278..6297
                     /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 catcatcacg tgaattcgct cgagatggtc tcccactcag agctgaggaa gcttttctac
      181 tcagcagatg ctgtgtgttt tgatgttgac agcacggtca tcagagaaga aggaatcgat
      241 gagctagcca aaatctgtgg cgttgaggac gcggtgtcag aaatgacacg gcgagccatg
      301 ggcggggcag tgcctttcaa agctgctctc acagagcgct tagccctcat ccagccctcc
      361 agggagcagg tgcagagact catagcagag caacccccac acctgacccc cggcataagg
      421 gagctggtaa gtcgcctaca ggagcgaaat gttcaggttt tcctaatatc tggtggcttt
      481 aggagtattg tagagcatgt tgcttcaaag ctcaatatcc cagcaaccaa tgtatttgcc
      541 aataggctga aattctactt taacggtgaa tatgcaggtt ttgatgagac gcagccaaca
      601 gctgaatctg gtggaaaagg aaaagtgatt aaacttttaa aggaaaaatt tcattttaag
      661 aaaataatca tgattggaga tggtgccaca gatatggaag cctgtcctcc tgctgatgct
      721 ttcattggat ttggaggaaa tgtgatcagg caacaagtca aggataacgc caaatggtat
      781 atcactgatt ttgtagagct gctgggagaa ctggaagaat aaggatccgg ctgctaacaa
      841 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
      901 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata
      961 tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg
     1021 acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt
     1081 tagcaattta actgtgataa actaccgcat taaagctagc ttatcgatga taagctgtca
     1141 aacatgagaa ttaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt
     1201 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     1261 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1321 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     1381 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     1441 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     1501 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     1561 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     1621 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1681 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1741 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     1801 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     1861 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     1921 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     1981 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2041 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     2101 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     2161 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     2221 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     2281 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     2341 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2401 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2461 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2521 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     2581 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2641 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2701 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2761 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     2821 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     2881 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     2941 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3001 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3061 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3121 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     3181 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     3241 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     3301 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     3361 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     3421 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     3481 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     3541 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     3601 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     3661 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     3721 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     3781 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     3841 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     3901 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     3961 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     4021 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac
     4081 gctgcccgag atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg
     4141 ccaagggttg gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg
     4201 agtggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc
     4261 catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat
     4321 gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca
     4381 atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg
     4441 tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg
     4501 agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag
     4561 cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     4621 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     4681 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     4741 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     4801 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     4861 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     4921 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4981 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     5041 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     5101 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     5161 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     5221 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     5281 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     5341 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     5401 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     5461 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     5521 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     5581 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     5641 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     5701 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     5761 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     5821 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     5881 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     5941 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     6001 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     6061 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     6121 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     6181 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     6241 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     6301 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     6361 a
//
