LOCUS       sequence-160844-        6724 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 6724)
  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 6724)
  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-GPD1
FEATURES             Location/Qualifiers
     source          1..6724
                     /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             115..132
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1236..1254)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1250..1297
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1535..1553)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1621..1725
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1726..1794,1795..2586)
                     /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(1944..1963)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2757..3345
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3246..3265
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3499..3516
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3531..3670
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3596..3615)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3756..3778
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3772..3963)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5275..6357)
                     /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     6319..6338
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6358..6435)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6641..6660
                     /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 gaaacatcat
      121 catcatcatc accagatggc tagcaagaaa gtctgcattg taggctccgg gaactggggc
      181 tcagccatcg ccaagatcgt gggtggcaat gcagcccagc tggcacagtt tgacccacgg
      241 gtgaccatgt gggtatttga ggaagacatt ggaggcaaaa agctgactga gatcatcaac
      301 acgcagcatg agaatgtcaa atacctgcca gggcacaagt tgcccccaaa tgtggtggct
      361 gtcccagatg tggtccaggc tgcagaggat gctgacatcc tgatctttgt ggtgccccat
      421 cagttcatcg gcaagatctg tgaccagctc aagggccatc tgaaggcaaa cgccactggc
      481 atatctctta ttaagggggt agacgagggc cccaatgggc tgaagctcat ctcggaagtg
      541 attggggagc gcctcggcat ccccatgagt gtgctgatgg gggccaacat tgccagcgag
      601 gtggctgatg agaagttctg tgagacaacc attggctgca aggacccggc ccagggacaa
      661 ctcctgaaag agctgatgca gacaccaaac ttccgtatca cagtggtgca agaggtggac
      721 acagtagaga tctgtggagc cttaaagaat gtagtggccg tgggggctgg cttctgtgat
      781 ggcctgggct ttggcgacaa caccaaggcg gcagtgatcc ggctgggact catggagatg
      841 atagccttcg ccaagctctt ctgcagtggc cctgtgtcct ctgccacctt cttggagagc
      901 tgtggtgttg ctgacctgat cactacctgc tatggagggc ggaaccggaa agtggctgag
      961 gcctttgcgc gtacaggaaa gtccattgag cagctggaga aagagttgct gaatgggcag
     1021 aaactgcagg ggcccgagac agcccgggag ctatacagca tcctccagca caagggcctg
     1081 gtagacaagt ttcccttgtt catggctgtg tacaaggtgt gctacgaggg ccagccagtg
     1141 ggtgaattca tccactgcct gcagaatcat ccagaacata tgtgaggatc cggctgctaa
     1201 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     1261 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     1321 atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg
     1381 gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg
     1441 cgttagcaat ttaactgtga taaactaccg cattaaagct agcttatcga tgataagctg
     1501 tcaaacatga gaattaattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     1561 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     1621 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     1681 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     1741 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     1801 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     1861 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     1921 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     1981 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     2041 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     2101 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     2161 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     2221 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
     2281 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     2341 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     2401 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     2461 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     2521 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     2581 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     2641 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     2701 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2761 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2821 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2881 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     2941 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3001 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3061 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3121 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3181 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3241 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3301 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3361 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3421 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3481 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3541 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     3601 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     3661 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3721 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3781 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     3841 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     3901 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     3961 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     4021 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     4081 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     4141 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     4201 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     4261 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     4321 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     4381 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gccaggaccc
     4441 aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga tggatatgtt
     4501 ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg ctccaattct
     4561 tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg
     4621 ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc gcctacaatc
     4681 catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac gatcagcggt
     4741 ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg tccctgatgg
     4801 tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat gccgccggaa
     4861 gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc cagcaagacg
     4921 tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     4981 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     5041 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     5101 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     5161 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     5221 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     5281 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5341 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     5401 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     5461 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     5521 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     5581 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     5641 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     5701 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     5761 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     5821 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     5881 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     5941 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     6001 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     6061 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     6121 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     6181 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     6241 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     6301 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     6361 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     6421 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     6481 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     6541 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     6601 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     6661 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     6721 tcga
//
