LOCUS       sequence-215040-        7038 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of RAPGEF4_DEP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7038)
  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 7038)
  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_RAPGEF4_DEP
FEATURES             Location/Qualifiers
     source          1..7038
                     /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      1233..1280
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2873..3328)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3010..3031)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3222..3241
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3370..3392)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3478..3618
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3533..3552
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3633..3650)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3804..4391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4562..5353,5354..5422))
                     /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     5185..5204
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5423..5527)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5595..5613
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5636..5713
                     /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             5714..6796
                     /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(5733..5752)
                     /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 cggccgtccc gtcggaaaaa attctgcgtg ctggcaagat tctgcgtaat gctattctgt
      961 cccgtgcccc gcacatgatt cgtgaccgta agtatcatct gaaaacctac cgtcagtgct
     1021 gtgtcggcac ggaactggtg gactggatga tgcagcaaac cccgtgcgtg catagccgta
     1081 cgcaggcagt tggcatgtgg caagtcctgc tggaagatgg tgtgctgaac cacgttgacc
     1141 aggaacatca ctttcaggat aagtacctgt tttatcgttt cctggatgac gaacacgaag
     1201 atgccccgct gccgaccgcg gccgcataat aaaccgatac aattaaaggc tccttttgga
     1261 gccttttttt tggagatttt caacgtgaaa aaattattat tcgcaattcc tttagttgtt
     1321 cctttctatt ctcactccgc tgaaactgtt gaaagttgtt tagcaaaacc ccatacagaa
     1381 aattcattta ctaacgtctg gaaagacgac aaaactttag atcgttacgc taactatgag
     1441 ggctgtctgt ggaatgctac aggcgttgta gtttgtactg gtgacgaaac tcagtgtcta
     1501 gctagagtgg cggtggctct ggttccggtg attttgatta tgaaaagatg gcaaacgcta
     1561 ataagggggc tatgaccgaa aatgccgatg aaaacgcgct acagtctgac gctaaaggca
     1621 aacttgattc tgtcgctact gattacggtg ctgctatcga tggtttcatt ggtgacgttt
     1681 ccggccttgc taatggtaat ggtgctactg gtgattttgc tggctctaat tcccaaatgg
     1741 ctcaagtcgg tgacggtgat aattcacctt taatgaataa tttccgtcaa tatttacctt
     1801 ccctccctca atcggttgaa tgtcgccctt ttgtctttgg cgctggtaaa ccatatgaat
     1861 tttctattga ttgtgacaaa ataaacttat tccgtggtgt ctttgcgttt cttttatatg
     1921 ttgccacctt tatgtatgta ttttctacgt ttgctaacat actgcgtaat aaggagtctt
     1981 aatcatgcca gttcttttgg ctagcgccgc cctatacctt gtctgcctcc ccgcgttgcg
     2041 tcgcggtgca tggagccggg ccacctcgac ctgaatggaa gccggcggca cctcgctaac
     2101 ggattcacca ctccaagaat tggagccaat caattcttgc ggagaactgt gaatgcgcaa
     2161 accaaccctt ggcagaacat atccatcgcg tccgccatct ccagcagccg cacgcggcgc
     2221 atctcgggca gcgttgggtc ctggccacgg gtgcgcatga tcgtgctcct gtcgttgagg
     2281 acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag
     2341 cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc
     2401 ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg
     2461 ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta
     2521 acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc
     2581 agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc
     2641 gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac
     2701 acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga
     2761 agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac
     2821 atctgtgaat cgcttcacga ccacgctgat gagctttacc gcaggatccg cgaaattgta
     2881 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     2941 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     3001 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     3061 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     3121 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     3181 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     3241 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     3301 gcgcttaatg cgccgctaca gggcgcgtcg cggatcctgc ctcgcgcgtt tcggtgatga
     3361 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     3421 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     3481 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     3541 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg
     3601 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3661 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3721 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3781 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3841 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccagggtttc
     3901 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3961 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4021 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4081 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4141 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4201 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4261 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4321 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4381 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4441 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4501 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4561 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4621 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4681 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4741 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4801 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4861 acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4921 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4981 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5041 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5101 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5161 gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5221 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5281 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5341 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5401 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5461 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5521 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     5581 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa ttcccgacac
     5641 catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     5701 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     5761 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     5821 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     5881 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     5941 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     6001 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     6061 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     6121 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     6181 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     6241 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     6301 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     6361 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     6421 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     6481 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac
     6541 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
     6601 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     6661 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     6721 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     6781 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcacaat
     6841 tctcatgttt gacagcttat catcgactgc acggtgcacc aatgcttctg gcgtcaggca
     6901 gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct
     6961 caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa
     7021 atattctgaa atgagctg
//
