LOCUS       sequence-160849-        6730 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 6730)
  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 6730)
  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-GPD1L
FEATURES             Location/Qualifiers
     source          1..6730
                     /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(1242..1260)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1256..1303
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1541..1559)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1627..1731
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1732..1800,1801..2592)
                     /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(1950..1969)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2763..3351
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3252..3271
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3505..3522
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3537..3676
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3602..3621)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3762..3784
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3778..3969)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5281..6363)
                     /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     6325..6344
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6364..6441)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6647..6666
                     /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 agcggcgccc ctgaaagtgt gcatcgtggg ctcggggaac
      181 tggggttcag ctgttgcaaa aataattggt aataatgtca agaaacttca gaaatttgcc
      241 tccacagtca agatgtgggt ctttgaagaa acagtgaatg gcagaaaact gacagacatc
      301 ataaataatg accatgaaaa tgtaaaatat cttcctggac acaagctgcc agaaaatgtg
      361 gttgccatgt caaatcttag cgaggctgtg caggatgcag acctgctggt gtttgtcatt
      421 ccccaccagt tcattcacag aatctgtgat gagatcactg ggagagtgcc caagaaagcg
      481 ctgggaatca ccctcatcaa gggcatagac gagggccccg aggggctgaa gctcatttct
      541 gacatcatcc gtgagaagat gggtattgac atcagtgtgc tgatgggagc caacattgcc
      601 aatgaggtgg ctgcagagaa gttctgtgag accaccatcg gcagcaaagt aatggagaac
      661 ggccttctct tcaaagaact tctgcagact ccaaattttc gaattaccgt ggttgatgat
      721 gcagacactg ttgaactctg tggtgcgctt aagaacatcg tagctgtggg agctgggttc
      781 tgcgacggcc tccgctgtgg agacaacacc aaagcggccg tcatccgcct gggactcatg
      841 gaaatgattg cttttgccag gatcttctgc aaaggccaag tgtctacagc caccttccta
      901 gagagctgcg gggtggccga cctgatcacc acctgttacg gagggcggaa ccgcagggtg
      961 gccgaggcct tcgccagaac tgggaagacc attgaagagt tggagaagga gatgctgaat
     1021 gggcaaaagc tccaaggacc gcagacttct gctgaagtgt accgcatcct caaacagaag
     1081 ggactactgg acaagtttcc attgtttact gcagtgtatc agatctgcta cgaaagcaga
     1141 ccagttcaag agatgttgtc ttgtcttcag agccatccag agcatacata aggatccggc
     1201 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     1261 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     1321 atccggatat cccgcaagag gcccggcagt accggcataa ccaagcctat gcctacagca
     1381 tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat acacggtgcc
     1441 tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagctagct tatcgatgat
     1501 aagctgtcaa acatgagaat taattcttga agacgaaagg gcctcgtgat acgcctattt
     1561 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     1621 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1681 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     1741 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     1801 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     1861 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     1921 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     1981 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     2041 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2101 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     2161 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2221 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     2281 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     2341 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     2401 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     2461 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     2521 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     2581 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     2641 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     2701 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2761 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2821 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2881 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     2941 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     3001 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3061 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3121 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3181 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3241 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3301 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3361 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3421 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3481 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     3541 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     3601 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     3661 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     3721 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     3781 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     3841 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     3901 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     3961 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     4021 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     4081 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     4141 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     4201 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     4261 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     4321 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     4381 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca
     4441 ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga
     4501 tatgttctgc caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc
     4561 aattcttgga gtggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg
     4621 gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct
     4681 acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc
     4741 agcggtccaa tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc
     4801 tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg
     4861 ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gaacgccagc
     4921 aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     4981 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     5041 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     5101 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     5161 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     5221 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     5281 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5341 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     5401 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     5461 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     5521 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     5581 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     5641 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     5701 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     5761 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     5821 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     5881 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     5941 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     6001 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     6061 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     6121 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     6181 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     6241 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     6301 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     6361 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     6421 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     6481 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     6541 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     6601 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     6661 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     6721 agaggatcga
//
