LOCUS       sequence-215951-        7764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of CACNB1_Guanylate-kin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7764)
  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 7764)
  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_CACNB1_Guanylate-kin
FEATURES             Location/Qualifiers
     source          1..7764
                     /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      1959..2006
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3599..4054)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3736..3757)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3948..3967
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4096..4118)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4204..4344
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4259..4278
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4359..4376)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4530..5117)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5288..6079,6080..6148))
                     /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     5911..5930
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6149..6253)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6321..6339
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6362..6439
                     /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             6440..7522
                     /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(6459..6478)
                     /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 cggcccgtcc gagtgattcc gacgttagcc tggaagaaga tcgtgaagcg ctgcgcaaag
      961 aagccgaacg tcaggcactg gctcaactgg aaaaagcgaa aaccaaaccg gtggcgtttg
     1021 ccgttcgcac gaacgtcggc tataatccgt caccgggtga cgaagttccg gtccagggcg
     1081 ttgccatcac ctttgaaccg aaagatttcc tgcatatcaa agaaaaatac aacaatgact
     1141 ggtggatcgg tcgtctggtg aaagaaggct gcgaagttgg tttcattccg tcaccggtga
     1201 aactggattc gctgcgcctg ctgcaggaac aaaaactgcg tcagaaccgc ctgggcagct
     1261 ctaaaagtgg tgataatagt tcctcatcgc tgggtgacgt ggttaccggt acgcgtcgcc
     1321 cgacgccgcc ggcaagcgct aaacagaaac aaaaatctac cgaacacgtg ccgccgtatg
     1381 atgtcgtgcc gtcaatgcgc ccgattatcc tggtcggccc gtcgctgaaa ggttacgaag
     1441 tgacggacat gatgcagaaa gcgctgtttg atttcctgaa acatcgtttt gacggccgca
     1501 tcagtattac ccgtgtgacg gcagatatta gtctggctaa acgctccgtt ctgaacaatc
     1561 cgagtaaaca cattatcatt gaacgttcca acacccgcag ctctctggca gaagtccagt
     1621 ccgaaatcga acgtattttc gaactggcac gtaccctgca actggtggcg ctggatgccg
     1681 acacgatcaa tcatccggca cagctgagca aaacctctct ggctccgatc attgtttata
     1741 tcaaaattac cagcccgaaa gtcctgcagc gtctgattaa atctcgcggt aaatcacagt
     1801 cgaaacatct gaacgttcaa atcgcggcct ctgaaaaact ggcgcaatgc ccgccggaaa
     1861 tgtttgatat cattctggac gaaaatcagc tggaagatgc gtgtgaacac ctggccgaat
     1921 atctggaagc atactggaaa gctgcggccg cataataaac cgatacaatt aaaggctcct
     1981 tttggagcct tttttttgga gattttcaac gtgaaaaaat tattattcgc aattccttta
     2041 gttgttcctt tctattctca ctccgctgaa actgttgaaa gttgtttagc aaaaccccat
     2101 acagaaaatt catttactaa cgtctggaaa gacgacaaaa ctttagatcg ttacgctaac
     2161 tatgagggct gtctgtggaa tgctacaggc gttgtagttt gtactggtga cgaaactcag
     2221 tgtctagcta gagtggcggt ggctctggtt ccggtgattt tgattatgaa aagatggcaa
     2281 acgctaataa gggggctatg accgaaaatg ccgatgaaaa cgcgctacag tctgacgcta
     2341 aaggcaaact tgattctgtc gctactgatt acggtgctgc tatcgatggt ttcattggtg
     2401 acgtttccgg ccttgctaat ggtaatggtg ctactggtga ttttgctggc tctaattccc
     2461 aaatggctca agtcggtgac ggtgataatt cacctttaat gaataatttc cgtcaatatt
     2521 taccttccct ccctcaatcg gttgaatgtc gcccttttgt ctttggcgct ggtaaaccat
     2581 atgaattttc tattgattgt gacaaaataa acttattccg tggtgtcttt gcgtttcttt
     2641 tatatgttgc cacctttatg tatgtatttt ctacgtttgc taacatactg cgtaataagg
     2701 agtcttaatc atgccagttc ttttggctag cgccgcccta taccttgtct gcctccccgc
     2761 gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga atggaagccg gcggcacctc
     2821 gctaacggat tcaccactcc aagaattgga gccaatcaat tcttgcggag aactgtgaat
     2881 gcgcaaacca acccttggca gaacatatcc atcgcgtccg ccatctccag cagccgcacg
     2941 cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt gctcctgtcg
     3001 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
     3061 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
     3121 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc
     3181 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct
     3241 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat
     3301 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc
     3361 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc
     3421 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt
     3481 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag
     3541 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag gatccgcgaa
     3601 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     3661 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     3721 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     3781 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     3841 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     3901 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     3961 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     4021 cccgccgcgc ttaatgcgcc gctacagggc gcgtcgcgga tcctgcctcg cgcgtttcgg
     4081 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     4141 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     4201 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg
     4261 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
     4321 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4381 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4441 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4501 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4561 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4621 ggtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4681 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4741 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4801 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4861 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4921 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     4981 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5041 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5101 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5161 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5221 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5281 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5341 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5401 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5461 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5521 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5581 tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5641 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5701 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5761 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5821 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5881 cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa
     5941 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6001 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6061 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6121 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6181 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6241 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     6301 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattcc
     6361 cgacaccatc gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     6421 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     6481 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     6541 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     6601 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     6661 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     6721 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     6781 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     6841 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     6901 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     6961 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     7021 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     7081 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     7141 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     7201 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
     7261 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     7321 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     7381 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     7441 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     7501 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     7561 cacaattctc atgtttgaca gcttatcatc gactgcacgg tgcaccaatg cttctggcgt
     7621 caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt
     7681 gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc
     7741 tggcaaatat tctgaaatga gctg
//
