LOCUS       sequence-163403-        6691 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 6691)
  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 6691)
  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-GAPDH
FEATURES             Location/Qualifiers
     source          1..6691
                     /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(1203..1221)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1217..1264
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1502..1520)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1588..1692
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1693..1761,1762..2553)
                     /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(1911..1930)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2724..3312
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3213..3232
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3466..3483
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3498..3637
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3563..3582)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3723..3745
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3739..3930)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5242..6324)
                     /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     6286..6305
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6325..6402)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6608..6627
                     /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 cgagatgggg aaggtgaagg tcggagtcaa cggatttggt
      181 cgtattgggc gcctggtcac cagggctgct tttaactctg gtaaagtgga tattgttgcc
      241 atcaatgacc ccttcattga cctcaactac atggtttaca tgttccaata tgattccacc
      301 catggcaaat tccatggcac cgtcaaggct gagaacggga agcttgtcat caatggaaat
      361 cccatcacca tcttccagga gcgagatccc tccaaaatca agtggggcga tgctggcgct
      421 gagtacgtcg tggagtccac tggcgtcttc accaccatgg agaaggctgg ggctcatttg
      481 caggggggag ccaaaagggt catcatctct gccccctctg ctgatgcccc catgttcgtc
      541 atgggtgtga accatgagaa gtatgacaac agcctcaaga tcatcagcaa tgcctcctgc
      601 accaccaact gcttagcacc cctggccaag gtcatccatg acaactttgg tatcgtggaa
      661 ggactcatga ccacagtcca tgccatcact gccacccaga agactgtgga tggcccctcc
      721 gggaaactgt ggcgtgatgg ccgcggggct ctccagaaca tcatccctgc ctctactggc
      781 gctgccaagg ctgtgggcaa ggtcatccct gagctgaacg ggaagctcac tggcatggcc
      841 ttccgtgtcc ccactgccaa cgtgtcagtg gtggacctga cctgccgtct agaaaaacct
      901 gccaaatatg atgacatcaa gaaggtggtg aagcaggcgt cggagggccc cctcaagggc
      961 atcctgggct acactgagca ccaggtggtc tcctctgact tcaacagcga cacccactcc
     1021 tccacctttg acgctggggc tggcattgcc ctcaacgacc actttgtcaa gctcatttcc
     1081 tggtatgaca acgaatttgg ctacagcaac agggtggtgg acctcatggc ccacatggcc
     1141 tccaaggagt aaggatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     1201 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     1261 tttgctgaaa ggaggaacta tatccggata tcccgcaaga ggcccggcag taccggcata
     1321 accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg
     1381 ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat
     1441 taaagctagc ttatcgatga taagctgtca aacatgagaa ttaattcttg aagacgaaag
     1501 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     1561 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     1621 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     1681 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     1741 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     1801 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     1861 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     1921 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     1981 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     2041 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     2101 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     2161 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     2221 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     2281 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     2341 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     2401 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     2461 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     2521 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     2581 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     2641 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2701 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2761 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2821 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     2881 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2941 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3001 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3061 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3121 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3181 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3241 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3301 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3361 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3421 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3481 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3541 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     3601 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     3661 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3721 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     3781 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     3841 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     3901 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     3961 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     4021 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     4081 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     4141 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     4201 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     4261 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     4321 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     4381 gatcatgcgc acccgtggcc aggacccaac gctgcccgag atgcgccgcg tgcggctgct
     4441 ggagatggcg gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct
     4501 ccgcaagaat tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg
     4561 gcttccattc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag
     4621 acaaggtata gggcggcgcc tacaatccat gccaacccgt tccatgtgct cgccgaggcg
     4681 gcataaatcg ccgtgacgat cagcggtcca atgatcgaag ttaggctggt aagagccgcg
     4741 agcgatcctt gaagctgtcc ctgatggtcg tcatctacct gcctggacag catggcctgc
     4801 aacgcgggca tcccgatgcc gccggaagcg agaagaatca taatggggaa ggccatccag
     4861 cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt cggccgccat gccggcgata
     4921 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4981 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     5041 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     5101 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     5161 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     5221 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5281 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     5341 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     5401 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     5461 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     5521 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5581 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5641 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5701 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5761 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5821 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5881 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5941 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     6001 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     6061 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     6121 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     6181 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     6241 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     6301 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     6361 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     6421 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     6481 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6541 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     6601 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     6661 cggccacgat gcgtccggcg tagaggatcg a
//
