LOCUS       sequence-361533-        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     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             93..110
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1184..1202)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1198..1245
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1483..1501)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1569..1673
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1674..1742,1743..2534)
                     /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(1892..1911)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2705..3293
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3194..3213
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3447..3464
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3479..3618
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3544..3563)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3704..3726
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3720..3911)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5223..6305)
                     /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     6267..6286
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6306..6383)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6589..6608
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaaact ttaagaagga gatatacata tgcatcatca tcatcatcac gtgaattcgc
      121 tcgagatggg gaaggtgaag gtcggagtca acggatttgg tcgtattggg cgcctggtca
      181 ccagggctgc ttttaactct ggtaaagtgg atattgttgc catcaatgac cccttcattg
      241 acctcaacta catggtttac atgttccaat atgattccac ccatggcaaa ttccatggca
      301 ccgtcaaggc tgagaacggg aagcttgtca tcaatggaaa tcccatcacc atcttccagg
      361 agcgagatcc ctccaaaatc aagtggggcg atgctggcgc tgagtacgtc gtggagtcca
      421 ctggcgtctt caccaccatg gagaaggctg gggctcattt gcagggggga gccaaaaggg
      481 tcatcatctc tgccccctct gctgatgccc ccatgttcgt catgggtgtg aaccatgaga
      541 agtatgacaa cagcctcaag atcatcagca atgcctcctg caccaccaac tgcttagcac
      601 ccctggccaa ggtcatccat gacaactttg gtatcgtgga aggactcatg accacagtcc
      661 atgccatcac tgccacccag aagactgtgg atggcccctc cgggaaactg tggcgtgatg
      721 gccgcggggc tctccagaac atcatccctg cctctactgg cgctgccaag gctgtgggca
      781 aggtcatccc tgagctgaac gggaagctca ctggcatggc cttccgtgtc cccactgcca
      841 acgtgtcagt ggtggacctg acctgccgtc tagaaaaacc tgccaaatat gatgacatca
      901 agaaggtggt gaagcaggcg tcggagggcc ccctcaaggg catcctgggc tacactgagc
      961 accaggtggt ctcctctgac ttcaacagcg acacccactc ctccaccttt gacgctgggg
     1021 ctggcattgc cctcaacgac cactttgtca agctcatttc ctggtatgac aacgaatttg
     1081 gctacagcaa cagggtggtg gacctcatgg cccacatggc ctccaaggag taaggatccg
     1141 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1201 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     1261 atatccggat atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag
     1321 catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg
     1381 cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctag cttatcgatg
     1441 ataagctgtc aaacatgaga attaattctt gaagacgaaa gggcctcgtg atacgcctat
     1501 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     1561 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1621 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     1681 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     1741 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     1801 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     1861 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     1921 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1981 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     2041 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     2101 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     2161 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     2221 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     2281 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     2341 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     2401 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     2461 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     2521 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     2581 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     2641 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     2701 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     2761 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     2821 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     2881 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     2941 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3001 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3061 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3121 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3181 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3241 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3301 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3361 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3421 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     3481 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     3541 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     3601 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     3661 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     3721 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     3781 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     3841 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     3901 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     3961 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4021 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     4081 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4141 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     4201 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     4261 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     4321 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc
     4381 caggacccaa cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg
     4441 gatatgttct gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct
     4501 ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg
     4561 tggcccggct ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc
     4621 ctacaatcca tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga
     4681 tcagcggtcc aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc
     4741 cctgatggtc gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc
     4801 cgccggaagc gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca
     4861 gcaagacgta gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga
     4921 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4981 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     5041 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     5101 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     5161 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     5221 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5281 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     5341 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     5401 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     5461 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     5521 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     5581 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     5641 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5701 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     5761 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5821 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5881 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5941 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     6001 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     6061 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     6121 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     6181 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     6241 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     6301 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     6361 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     6421 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     6481 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     6541 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     6601 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     6661 gtagaggatc gagatctcga tcccgcgaaa t
//
