LOCUS       sequence-342404-        8044 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8044)
  AUTHORS   Banko MR, Allen JJ, Schaffer BE, Wilker EW, Tsou P, White JL, Villen
            J, Wang B, Kim SR, Sakamoto K, Gygi SP, Cantley LC, Yaffe MB, Shokat
            KM, Brunet A
  TITLE     Chemical Genetic Screen for AMPKalpha2 Substrates Uncovers a Network
            of Proteins Involved in Mitosis.
  JOURNAL   Mol Cell. 2011 Dec 23;44(6):878-92. Epub 2011 Dec 1.
  PUBMED    22137581
REFERENCE   2  (bases 1 to 8044)
  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"; journalName: "Mol Cell.";
            date: "2011-12-23"; volume: "44(6)"; pages: "878-92. Epub 2011 Dec 
            1"
COMMENT     Sequence Label: pGEX-PPP1R12A wt
FEATURES             Location/Qualifiers
     source          1..8044
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..17
                     /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             40..693
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     651..673
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             700..717
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(3876..3898)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4043..4061)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4129..4233
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4234..4302,4303..5094)
                     /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(4452..4471)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5265..5853
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5754..5773
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6007..6024
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6097..6174
                     /gene="lacI"
                     /label=lacI promoter
     CDS             6175..7257
                     /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(6194..6213)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(7306..7311,7312..7329,7330..7336)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7344..7360
                     /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     7368..7384
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7368..7384
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             7380..7907
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(7400..7417)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7400..7416)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7409..7431)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(8009..8014,8015..8030,8031..8037)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
ORIGIN
        1 ttgtgagcgg ataacaattt cacacaggaa acagtattca tgtcccctat actaggttat
       61 tggaaaatta agggccttgt gcaacccact cgacttcttt tggaatatct tgaagaaaaa
      121 tatgaagagc atttgtatga gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa
      181 ttgggtttgg agtttcccaa tcttccttat tatattgatg gtgatgttaa attaacacag
      241 tctatggcca tcatacgtta tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa
      301 gagcgtgcag agatttcaat gcttgaagga gcggttttgg atattagata cggtgtttcg
      361 agaattgcat atagtaaaga ctttgaaact ctcaaagttg attttcttag caagctacct
      421 gaaatgctga aaatgttcga agatcgttta tgtcataaaa catatttaaa tggtgatcat
      481 gtaacccatc ctgacttcat gttgtatgac gctcttgatg ttgttttata catggaccca
      541 atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa aacgtattga agctatccca
      601 caaattgata agtacttgaa atccagcaag tatatagcat ggcctttgca gggctggcaa
      661 gccacgtttg gtggtggcga ccatcctcca aaatcggatc tggttccgcg tggatccccg
      721 aattcgcaga tggcggacgc gaagcagaag cggaacgagc agctgaaacg ctggatcggc
      781 tccgagacgg acctcgagcc tccggtggtg aagcgccaga agaccaaggt gaagttcgac
      841 gatggcgccg tcttcctggc tgcttgctcc agcggcgaca cggacgaggt cctcaagctg
      901 ctgcaccgcg gcgccgacat caattacgcc aatgtggacg gactcactgc cctgcaccag
      961 gcttgcattg atgacaatgt tgatatggtg aagtttctgg tagaaaatgg agcaaatatt
     1021 aatcaacctg ataatgaagg ctggatacca ctacatgcag cagcttcctg tggatatctt
     1081 gatattgcag agtttttgat tggtcaagga gcacatgtag gggctgtcaa cagtgaagga
     1141 gatacacctt tagatattgc ggaggaggag gcaatggaag agctacttca aaatgaagtt
     1201 aatcggcaag gggttgatat agaagcagct cgaaaggaag aagaacggat catgcttaga
     1261 gatgccaggc agtggctaaa tagtggtcat ataaatgatg tccggcatgc aaaatctgga
     1321 ggtacagcac ttcacgttgc agctgctaaa ggctatacgg aagttttaaa acttttaata
     1381 caggcaggct atgatgttaa tattaaagac tatgatggct ggacacctct tcatgctgca
     1441 gctcattggg gtaaagaaga agcatgtcga attttagtgg acaatctgtg tgatatggag
     1501 atggtcaaca aagtgggcca aacagccttt gatgtagcag atgaagacat tttaggatat
     1561 ttagaagagt tgcaaaagaa acaaaatctg ctccatagtg aaaaacggga caagaaatct
     1621 ccactaattg aatcaacagc aaatatggac aataatcagt cacagaagac ctttaaaaac
     1681 aaagagacgt tgattattga accagagaaa aatgcatccc gtattgaatc tctggaacaa
     1741 gaaaaggttg atgaagaaga agaaggaaag aaggatgagt ctagctgctc tagtgaagaa
     1801 gatgaggaag atgactcgga atcagaagct gaaacagata agacaaaacc cctggcttct
     1861 gtaactaatg ccaacacttc tagtacacaa gcagctcctg tagctgttac aacacctact
     1921 gtgtcatcag gtcaagcaac acctacatca cctattaaaa agtttccaac cacagctaca
     1981 aaaatttctc ccaaagaaga agagagaaaa gatgagtctc ctgcaacttg gaggttagga
     2041 cttagaaaga cgggcagcta tggtgcactt gctgaaatca cagcatctaa agagggtcag
     2101 aaagaaaaag atactgcagg tgttacacgt tcagcttcaa gtcccagact ttcctcctct
     2161 ttggataata aagaaaagga gaaagatagt aaaggaacta ggcttgcata tgttgcacct
     2221 acaataccaa gacgactagc cagtacatct gacattgaag agaaagaaaa cagagattct
     2281 tcaagtttgc gaacaagtag ttcatataca aggagaaaat gggaagatga tcttaaaaaa
     2341 aatagctcag ttaatgaagg atcaacgtat cataaaagtt gctcctttgg tagaagacaa
     2401 gatgatttga ttagttctag tgttccaagc accacatcaa caccaacagt tacctctgca
     2461 gctgggcttc agaaaagcct gctttccagc acaagcacta ctacaaagat tacaacgggt
     2521 tcttcctcag caggcacaca aagcagtacc tcaaatcgtt tgtgggctga ggatagtact
     2581 gagaaagaaa aggacagtgt tcctacggca gtgaccattc ctgttgctcc aactgttgta
     2641 aatgctgcag cttctaccac aaccctgact acaactactg ctggcactgt ctcctccaca
     2701 acagaggtca gggagagacg cagatcatac ctcactcctg ttagggatga agagtctgaa
     2761 tcccaaagaa aagcaagatc tagacaagca agacaatcta gaagatcaac acagggagtg
     2821 acattaactg atcttcaaga agctgagaaa acaataggaa gaagtcgttc tacccgaacc
     2881 agagaacaag aaaatgaaga aaaagaaaaa gaggaaaaag agaaacaaga taaagagaaa
     2941 caagaagaaa agaaggagtc agaaacatct agagaagatg aatataaaca aaagtactcc
     3001 agaacgtatg atgagactta ccagcgttat aggccagtat caacttcaag ttcaaccact
     3061 ccatcctctt cactttctac tatgagcagt tcactgtatg cttcaagtca actaaacagg
     3121 ccaaatagtc ttgtaggcat aacttctgct tactccagag gaataacaaa agaaaatgaa
     3181 agagagggag aaaaaagaga agaggagaaa gaaggagaag ataaatcaca acctaaatca
     3241 atcagagaac gacgacgacc aagagagaaa agaagatcta caggagtttc attttggaca
     3301 caagatagtg atgaaaatga acaagaacaa caatcagaca cagaagaggg atccaataag
     3361 aaagaaactc agacggattc catttctaga tatgaaacca gttctacatc agctggtgat
     3421 cgatatgatt ccttgctggg tcgctctgga tcatacagtt acttagaaga aagaaaacct
     3481 tacagcagca ggctagaaaa ggatgactca actgacttta aaaagcttta tgaacaaatt
     3541 ctagctgaaa atgaaaagct gaaggcacag ctacatgata caaatatgga actaacagat
     3601 cttaaattac agttggaaaa ggccacccag agacaagaaa gatttgctga tagatcactg
     3661 ttggaaatgg aaaaaaggga acgaagagct ctagaaagaa gaatatctga aatggaagaa
     3721 gagctcaaaa tgttaccaga cctaaaagca gacaaccaga ggctaaagga tgaaaatggg
     3781 gccttgatca gagttataag caaactttcc aaataacggc cgcatcgtga ctgactgacg
     3841 atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     3901 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     3961 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     4021 tataattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     4081 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4141 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4201 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     4261 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     4321 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     4381 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     4441 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
     4501 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     4561 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     4621 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     4681 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     4741 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     4801 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     4861 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     4921 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     4981 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     5041 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5101 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5161 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5221 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5281 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     5341 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5401 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5461 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5521 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5581 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5641 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5701 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5761 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5821 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5881 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     5941 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     6001 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     6061 acgcatctgt gcggtatttc acaccgcata aattccgaca ccatcgaatg gcgcaaaacc
     6121 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     6181 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     6241 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     6301 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     6361 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     6421 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     6481 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     6541 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     6601 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     6661 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     6721 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     6781 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     6841 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     6901 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     6961 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     7021 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     7081 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     7141 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     7201 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     7261 caacgcaatt aatgtaagtt agctcactca ttaggcaccc caggctttac actttatgct
     7321 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     7381 tgaccatgat tacggattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     7441 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     7501 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgct
     7561 ttgcctggtt tccggcacca gaagcggtgc cggaaagctg gctggagtgc gatcttcctg
     7621 aggccgatac tgtcgtcgtc ccctcaaact ggcagatgca cggttacgat gcgcccatct
     7681 acaccaacgt gacctatccc attacggtca atccgccgtt tgttcccacg gagaatccga
     7741 cgggttgtta ctcgctcaca tttaatgttg atgaaagctg gctacaggaa ggccagacgc
     7801 gaattatttt tgatggcgtt ggaattagct tatcgactgc acggtgcacc aatgcttctg
     7861 gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat
     7921 tcgtgtcgct caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg
     7981 gttctggcaa atattctgaa atgagctgtt gacaattaat catcggctcg tataatgtgt
     8041 ggaa
//
