LOCUS       sequence-215942-        7491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of PRKACA_C1+C2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7491)
  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 7491)
  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_PRKACA_C1+C2
FEATURES             Location/Qualifiers
     source          1..7491
                     /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      1686..1733
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3326..3781)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3463..3484)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3675..3694
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3823..3845)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3931..4071
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3986..4005
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4086..4103)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4257..4844)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5015..5806,5807..5875))
                     /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     5638..5657
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5876..5980)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6048..6066
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6089..6166
                     /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             6167..7249
                     /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(6186..6205)
                     /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 cggcctttga acgtattaaa accctgggca cgggtagttt cggtcgcgtc atgctggtga
      961 aacataaaga aaccggcaat cactatgcga tgaaaatcct ggataaacag aaagtggtta
     1021 aactgaaaca aatcgaacat acgctgaacg aaaaacgtat cctgcaggcc gtcaactttc
     1081 cgttcctggt gaaactggaa ttcagtttca aagataactc caacctgtat atggttatgg
     1141 aatacgtccc gggcggtgaa atgtttagcc atctgcgtcg cattggtcgt ttttctgaac
     1201 cgcacgcacg cttctatgcg gcccagattg tgctgacctt tgaatatctg cactccctgg
     1261 atctgatcta ccgcgacctg aaaccggaaa atctgctgat tgatcagcaa ggttacatcc
     1321 aagtgaccga ctttggcttc gcaaaacgtg ttaaaggtcg tacctggacg ctgtgcggta
     1381 cgccggaata tctggctccg gaaattatcc tgagcaaagg ctacaacaaa gcggttgatt
     1441 ggtgggccct gggtgtcctg atttatgaaa tggcagctgg ctacccgccg tttttcgcgg
     1501 accagccgat tcaaatctat gaaaaaatcg tgtcaggcaa agttcgtttt ccgtcgcatt
     1561 tcagctctga tctgaaagac ctgctgcgta atctgctgca ggtcgatctg accaaacgct
     1621 ttggcaacct gaaaaatggt gttaacgaca tcaaaaacca caaatggttc gcggccgcat
     1681 aataaaccga tacaattaaa ggctcctttt ggagcctttt ttttggagat tttcaacgtg
     1741 aaaaaattat tattcgcaat tcctttagtt gttcctttct attctcactc cgctgaaact
     1801 gttgaaagtt gtttagcaaa accccataca gaaaattcat ttactaacgt ctggaaagac
     1861 gacaaaactt tagatcgtta cgctaactat gagggctgtc tgtggaatgc tacaggcgtt
     1921 gtagtttgta ctggtgacga aactcagtgt ctagctagag tggcggtggc tctggttccg
     1981 gtgattttga ttatgaaaag atggcaaacg ctaataaggg ggctatgacc gaaaatgccg
     2041 atgaaaacgc gctacagtct gacgctaaag gcaaacttga ttctgtcgct actgattacg
     2101 gtgctgctat cgatggtttc attggtgacg tttccggcct tgctaatggt aatggtgcta
     2161 ctggtgattt tgctggctct aattcccaaa tggctcaagt cggtgacggt gataattcac
     2221 ctttaatgaa taatttccgt caatatttac cttccctccc tcaatcggtt gaatgtcgcc
     2281 cttttgtctt tggcgctggt aaaccatatg aattttctat tgattgtgac aaaataaact
     2341 tattccgtgg tgtctttgcg tttcttttat atgttgccac ctttatgtat gtattttcta
     2401 cgtttgctaa catactgcgt aataaggagt cttaatcatg ccagttcttt tggctagcgc
     2461 cgccctatac cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc
     2521 gacctgaatg gaagccggcg gcacctcgct aacggattca ccactccaag aattggagcc
     2581 aatcaattct tgcggagaac tgtgaatgcg caaaccaacc cttggcagaa catatccatc
     2641 gcgtccgcca tctccagcag ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca
     2701 cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt
     2761 actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa
     2821 acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg
     2881 gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc
     2941 tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg
     3001 atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt
     3061 aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt
     3121 atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa
     3181 aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa
     3241 ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct
     3301 gatgagcttt accgcaggat ccgcgaaatt gtaagcgtta atattttgtt aaaattcgcg
     3361 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     3421 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     3481 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     3541 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     3601 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     3661 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     3721 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     3781 tcgcggatcc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     3841 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     3901 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     3961 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     4021 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     4081 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4141 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4201 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4261 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4321 aacccgacag gactataaag ataccagggt ttccccctgg aagctccctc gtgcgctctc
     4381 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4441 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4501 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4561 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4621 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4681 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4741 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4801 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4861 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4921 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4981 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5041 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5101 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5161 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5221 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5281 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg
     5341 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5401 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5461 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5521 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5581 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata
     5641 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5701 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5761 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5821 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5881 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5941 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6001 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     6061 cgaggccctt tcgtcttcaa gaattcccga caccatcgaa tggtgcaaaa cctttcgcgg
     6121 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
     6181 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     6241 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     6301 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     6361 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     6421 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     6481 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     6541 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     6601 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     6661 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     6721 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     6781 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     6841 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     6901 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     6961 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     7021 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     7081 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     7141 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     7201 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     7261 ttaatgtaag ttagctcact cattaggcac aattctcatg tttgacagct tatcatcgac
     7321 tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg
     7381 caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg
     7441 ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct g
//
