LOCUS       sequence-477347-        6988 bp    DNA     circular SYN 30-JUN-2025
DEFINITION  To express in E. coli KGA F7 with 33 residues mutated to alanine at 
            the interface for interacting with PDZD8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6988)
  AUTHORS   Li M, Wang Y, Wei X, Cai WF, Wu J, Zhu M, Wang Y, Liu YH, Xiong J, 
            Qu Q, Chen Y, Tian X, Yao L, Xie R, Li X, Chen S, Huang X, Zhang C, 
            Xie C, Wu Y, Xu Z, Zhang B, Jiang B, Wang ZC, Li Q, Li G, Lin SY, Yu
            L, Piao HL, Deng X, Han J, Zhang CS, Lin SC
  TITLE     AMPK targets PDZD8 to trigger carbon source shift from glucose to 
            glutamine.
  JOURNAL   Cell Res. 2024 Jun 19. doi: 10.1038/s41422-024-00985-6.
  PUBMED    38898113
REFERENCE   2  (bases 1 to 6988)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 30, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Res. 
            2024 Jun 19. doi: 10.1038/s41422-024-00985-6."
COMMENT     Sequence Label: pGEX KGA F7
FEATURES             Location/Qualifiers
     source          1..6988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(3038..3060)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3205..3223)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3291..3395
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3396..3464,3465..4256)
                     /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(3614..3633)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4427..5015
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4916..4935
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5169..5186
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5259..5336
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5337..6419
                     /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     complement(5356..5375)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6468..6473,6474..6491,6492..6498)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6506..6522
                     /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)."
     primer_bind     6530..6546
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6530..6546
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6542..6988,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6562..6579)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6562..6578)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6571..6593)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatcgagaga attcggatcc atgatgcggc
      961 tgcgaggctc ggggatgctg cgggacctgc tcctgcggtc gcccgcagga gtatcggcga
     1021 ctctgcggcg ggcacagccc ttggtcaccc tgtgccggcg tccccgaggc gggggacggc
     1081 cggccgcggg cccggctgcc gccgcgcgac tccacccgtg gtggggcggg ggcggctggc
     1141 cggcggagcc cctcgcgcgg ggcctgtcca gctctccttc ggagatcttg caggagctgg
     1201 gcaaggggag cacgcatccg cagcccgggg tgtcgccacc cgctgccccg gcggcgcccg
     1261 gccccaagga cggccccggg gagacggacg cgtttggcaa cagcgagggc aaagagctgg
     1321 tggcctcagg tgaaaataaa ataaaacagg gtctgttagc tagcgcggca gatgcgctgt
     1381 tcgctacaat tgctgaagca gcagcgaaaa tacctgttca taaatttatt acagcactca
     1441 aatctacagg attgcgaacg tctgatccca ggttggcagc gtgtatggct atgttaagat
     1501 taactcttgc aacaacatca gatggtgtca tgctagacaa agatcttttt aaaaaatgtg
     1561 ttcagagcaa cattgctttg ttggcagcag catttgcaag agcgtttgtg attcctgcct
     1621 ttatggcttt tacctcagcc attgatgagt tatatgaaag tgctaaaaag cagtctggag
     1681 gaaaggttgc agattatatt cctcaactgg ccaaattcag tcccgatttg tggggtgtgt
     1741 ctgtttgtac agtagatgga cagaggcatt ctactggaga taccaaagtt cccttctgtc
     1801 ttcagtcctg tgtaaaacct ttgaaatatg ccattgctgt taatgatctt ggaactgaat
     1861 atgtgcatcg atatgttgga aaagagccga gtggactaag attcaacaaa ctatttttga
     1921 atgaagatga taaaccacat aatcctatgg taaatgctgg agcaattgtt gtgacttcac
     1981 taataaagca aggagtaaat aatgctgaaa aatttgacta tgtcatgcag tttttgaata
     2041 agatggctgg taatgaatat gttggattca gtaatgcaac gtttcagtct gaaagagaaa
     2101 gtggagatcg aaattttgca ataggatatt acttaaaaga aaagaagtgt tttccagaag
     2161 gcacagacat ggttggtata ttagacttct acttccagct gtgctccatt gaagtgactt
     2221 gtgaatcagc cagtgtgatg gctgcgacac tggctaatgg tggtgcctgc ccaattactg
     2281 ctgcagcagc actgagcgct gaagcagttc gaaatacatt gagtttgatg cattcctgtg
     2341 gcatgtatga cttctcaggg cagtttgctt tccatgttgg tcttcctgca aaatctggag
     2401 ttgctggggg cattctttta gttgtcccca atgttatggg tatgatgtgc tggtctcctc
     2461 ctctggataa gatgggcaac agtgttaagg gaattcactt ttgtcacgat cttgtttctc
     2521 tgtgtaattt ccataactat gataatttgg cagccgctgc agcaaaagct gatcctcgag
     2581 cagcaggtgg tgatcaaagg gtaaagtcag tgataaatct tttgtttgct gcatatactg
     2641 gagatgtgtc tgcacttcga agatttgctt tgtcagctat ggacatggaa cagcgggact
     2701 atgattctag aacagcactc catgtagctg ctgcagaggg tcatgttgaa gttgttaaat
     2761 ttttgctgga agcctgcaaa gtaaaccctt tccccaagga caggtggaat aacactccca
     2821 tggatgaagc actgcacttt ggacaccatg atgtatttaa aattctccaa gaataccaag
     2881 tccagtacac acctcaagga gattctgaca acgggaagga aaatcaaaca gtccataaga
     2941 atcttgatgg attgttgctc gagccatgga aggtcgagcg gccgcatcgt gactgactga
     3001 cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     3061 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     3121 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     3181 tgtataattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     3241 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3301 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3361 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3421 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3481 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3541 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3601 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     3661 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3721 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3781 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3841 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3901 aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
     3961 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     4021 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4081 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4141 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4201 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4261 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4321 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4381 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4441 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4501 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4561 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4621 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4681 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4741 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4801 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4861 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4921 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4981 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5041 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     5101 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     5161 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     5221 ttacgcatct gtgcggtatt tcacaccgca taaattccga caccatcgaa tggcgcaaaa
     5281 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     5341 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     5401 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     5461 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     5521 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     5581 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     5641 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     5701 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     5761 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     5821 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     5881 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     5941 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     6001 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     6061 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     6121 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     6181 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     6241 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     6301 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     6361 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     6421 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cccaggcttt acactttatg
     6481 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     6541 tatgaccatg attacggatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     6601 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     6661 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     6721 ctttgcctgg tttccggcac cagaagcggt gccggaaagc tggctggagt gcgatcttcc
     6781 tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat
     6841 ctacaccaac gtgacctatc ccattacggt caatccgccg tttgttccca cggagaatcc
     6901 gacgggttgt tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac
     6961 gcgaattatt tttgatggcg ttggaatt
//
