LOCUS       sequence-212752-        7635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of AKT1_Pkinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7635)
  AUTHORS   Teyra J, Kelil A, Jain S, Helmy M, Jajodia R, Hooda Y, Gu J, D'Cruz 
            AA, Nicholson SE, Min J, Sudol M, Kim PM, Bader GD, Sidhu SS
  TITLE     Large-scale survey and database of high affinity ligands for peptide
            recognition modules.
  JOURNAL   Mol Syst Biol. 2020 Dec;16(12):e9310. doi: 10.15252/msb.20199310.
  PUBMED    33438817
REFERENCE   2  (bases 1 to 7635)
  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 Syst 
            Biol."; date: "2020-12"; volume: "16(12):e9310. doi"; pages: " 
            10.15252/msb.20199310"
COMMENT     Sequence Label: pHH0103_AKT1_Pkinase
FEATURES             Location/Qualifiers
     source          1..7635
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             138..155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             171..824
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     782..804
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             831..854
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             867..884
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      1830..1877
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3470..3925)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3607..3628)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3819..3838
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3967..3989)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4075..4215
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4130..4149
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4230..4247)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4401..4988)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5159..5950,5951..6019))
                     /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     5782..5801
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6020..6124)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6192..6210
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6233..6310
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             6311..7393
                     /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(6330..6349)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga
      121 ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt atgtccccta
      181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
      841 tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc gcggcccagc
      901 cggcccgtgt tacgatgaat gaattcgagt acctgaaact gctgggcaag ggcacctttg
      961 gtaaagtaat cctggtcaaa gaaaaagcga ccggccgcta ctacgccatg aagatcctga
     1021 agaaagaagt aattgtagct aaagacgaag ttgcgcacac cctgacggaa aaccgtgtgc
     1081 tgcaaaactc ccgtcatcct ttcctgaccg ccctgaaata ctcctttcag acccacgatc
     1141 gtctgtgctt cgttatggaa tatgcgaacg gcggtgaact gtttttccat ctgtctcgtg
     1201 aacgcgtttt ctccgaggac cgtgcacgct tctatggtgc agagatcgta tctgctctgg
     1261 actacctgca tagcgagaaa aacgtggtgt accgcgatct gaaactggag aacctgatgc
     1321 tggacaagga cggtcacatc aaaatcaccg atttcggtct gtgcaaggaa ggtattaaag
     1381 atggcgcaac tatgaaaact ttttgtggca ccccggaata tctggcgccg gaagttctgg
     1441 aagataacga ttatggtcgt gccgtcgact ggtggggcct gggcgttgtg atgtatgaaa
     1501 tgatgtgtgg ccgtctgccg ttctacaacc aagaccacga aaaactgttt gaactgattc
     1561 tgatggaaga gattcgtttc ccgcgcactc tgggtccgga agctaaatct ctgctgtccg
     1621 gtctgctgaa aaaagaccca aaacagcgtc tgggcggtgg tagcgaagat gctaaagaaa
     1681 tcatgcagca ccgctttttc gcgggtatcg tttggcagca cgtttacgag aaaaaactga
     1741 gcccgccatt caaaccgcag gtgacctctg aaaccgacac tcgttacttc gatgaagaat
     1801 tcactgcaca gatggcggcc gcataataaa ccgatacaat taaaggctcc ttttggagcc
     1861 ttttttttgg agattttcaa cgtgaaaaaa ttattattcg caattccttt agttgttcct
     1921 ttctattctc actccgctga aactgttgaa agttgtttag caaaacccca tacagaaaat
     1981 tcatttacta acgtctggaa agacgacaaa actttagatc gttacgctaa ctatgagggc
     2041 tgtctgtgga atgctacagg cgttgtagtt tgtactggtg acgaaactca gtgtctagct
     2101 agagtggcgg tggctctggt tccggtgatt ttgattatga aaagatggca aacgctaata
     2161 agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct aaaggcaaac
     2221 ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt gacgtttccg
     2281 gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc caaatggctc
     2341 aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat ttaccttccc
     2401 tccctcaatc ggttgaatgt cgcccttttg tctttggcgc tggtaaacca tatgaatttt
     2461 ctattgattg tgacaaaata aacttattcc gtggtgtctt tgcgtttctt ttatatgttg
     2521 ccacctttat gtatgtattt tctacgtttg ctaacatact gcgtaataag gagtcttaat
     2581 catgccagtt cttttggcta gcgccgccct ataccttgtc tgcctccccg cgttgcgtcg
     2641 cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct cgctaacgga
     2701 ttcaccactc caagaattgg agccaatcaa ttcttgcgga gaactgtgaa tgcgcaaacc
     2761 aacccttggc agaacatatc catcgcgtcc gccatctcca gcagccgcac gcggcgcatc
     2821 tcgggcagcg ttgggtcctg gccacgggtg cgcatgatcg tgctcctgtc gttgaggacc
     2881 cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga
     2941 acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc
     3001 ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc
     3061 cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg
     3121 aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt
     3181 tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg
     3241 agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg
     3301 gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc
     3361 cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc
     3421 tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca ggatccgcga aattgtaagc
     3481 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3541 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3601 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3661 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     3721 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3781 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3841 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3901 cttaatgcgc cgctacaggg cgcgtcgcgg atcctgcctc gcgcgtttcg gtgatgacgg
     3961 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     4021 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     4081 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     4141 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     4201 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4261 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4321 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4381 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4441 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca gggtttcccc
     4501 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4561 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4621 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4681 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4741 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4801 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     4861 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4921 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4981 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5041 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5101 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5161 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5221 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5281 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5341 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5401 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5461 ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5521 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5581 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5641 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5701 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5761 cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5821 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5881 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5941 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6001 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6061 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6121 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     6181 taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaattc ccgacaccat
     6241 cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     6301 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     6361 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     6421 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     6481 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     6541 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     6601 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     6661 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     6721 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     6781 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     6841 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
     6901 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
     6961 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
     7021 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
     7081 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
     7141 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     7201 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     7261 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     7321 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     7381 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcacaattct
     7441 catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc
     7501 atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa
     7561 ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata
     7621 ttctgaaatg agctg
//
