LOCUS       sequence-479453-        6775 bp    DNA     circular SYN 23-JUL-2025
DEFINITION  To express in E. coli GAC 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 6775)
  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 6775)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 23, 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 GAC F7
FEATURES             Location/Qualifiers
     source          1..6775
                     /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(2825..2847)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2992..3010)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3078..3182
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3183..3251,3252..4043)
                     /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(3401..3420)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4214..4802
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4703..4722
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4956..4973
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5046..5123
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5124..6206
                     /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(5143..5162)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6255..6260,6261..6278,6279..6285)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6293..6309
                     /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     6317..6333
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6317..6333
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6329..6775,1..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(6349..6366)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6349..6365)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6358..6380)
                     /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 cattcctttg gaccattgga ctatgaaagt ctccaacaag
     2641 aacttgcttt aaaagagaca gtatggaaaa aagtgtcacc tgagtcaaat gaggacatct
     2701 ctacaactgt agtatataga atggaaagtc tgggagagaa aagcctcgag ccatggaagg
     2761 tcgagcggcc gcatcgtgac tgactgacga tctgcctcgc gcgtttcggt gatgacggtg
     2821 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     2881 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     2941 tgacccagtc acgtagcgat agcggagtgt ataattcttg aagacgaaag ggcctcgtga
     3001 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     3061 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3121 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3181 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     3241 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     3301 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     3361 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     3421 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     3481 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     3541 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     3601 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     3661 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     3721 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3781 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3841 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     3901 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3961 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     4021 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     4081 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4141 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4201 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4261 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4321 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4381 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4441 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4501 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4561 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4621 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4681 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4741 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4801 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4861 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4921 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4981 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcataa
     5041 attccgacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     5101 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     5161 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     5221 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     5281 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     5341 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     5401 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     5461 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     5521 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     5581 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     5641 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     5701 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     5761 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     5821 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     5881 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
     5941 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     6001 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     6061 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     6121 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     6181 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat
     6241 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     6301 ggataacaat ttcacacagg aaacagctat gaccatgatt acggattcac tggccgtcgt
     6361 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     6421 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     6481 gttgcgcagc ctgaatggcg aatggcgctt tgcctggttt ccggcaccag aagcggtgcc
     6541 ggaaagctgg ctggagtgcg atcttcctga ggccgatact gtcgtcgtcc cctcaaactg
     6601 gcagatgcac ggttacgatg cgcccatcta caccaacgtg acctatccca ttacggtcaa
     6661 tccgccgttt gttcccacgg agaatccgac gggttgttac tcgctcacat ttaatgttga
     6721 tgaaagctgg ctacaggaag gccagacgcg aattattttt gatggcgttg gaatt
//
