LOCUS       sequence-192276-        6912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            ARHGEF4-1/1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6912)
  AUTHORS   Teyra J, Huang H, Jain S, Guan X, Dong A, Liu Y, Tempel W, Min J, 
            Tong Y, Kim PM, Bader GD, Sidhu SS
  TITLE     Comprehensive Analysis of the Human SH3 Domain Family Reveals a Wide
            Variety of Non-canonical Specificities.
  JOURNAL   Structure. 2017 Oct 3;25(10):1598-1610.e3. doi: 
            10.1016/j.str.2017.07.017. Epub 2017 Sep 7.
  PUBMED    28890361
REFERENCE   2  (bases 1 to 6912)
  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"; doi: 
            "10.1016/j.str.2017.07.017"; journalName: "Structure"; date: 
            "2017-10-3- 3"; volume: "25"; issue: "10"; pages: "1598-1610.e3"
COMMENT     Sequence Label: pHH0103_ARHGEF4-1/1
FEATURES             Location/Qualifiers
     source          1..6912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..23
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        46..123
                     /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             124..1206
                     /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(143..162)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1449..1454,1455..1470,1471..1477)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1485..1501
                     /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     1490..1512
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1586..1603
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1619..2272
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2230..2252
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2279..2302
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2315..2332
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2555..2602
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4195..4650)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4332..4353)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4544..4563
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4692..4714)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4800..4940
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4855..4874
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4955..4972)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5126..5713)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5884..6675,6676..6744))
                     /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     6507..6526
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6745..6849)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 taaaaatagg cgtatcacga ggccctttcg tcttcaagaa ttcccgacac catcgaatgg
       61 tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
      121 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
      181 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
      241 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
      301 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
      361 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
      421 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
      481 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
      541 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
      601 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
      661 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
      721 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
      781 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
      841 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
      901 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc
      961 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     1021 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     1081 cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc
     1141 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     1201 cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcacaat tctcatgttt
     1261 gacagcttat catcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa
     1321 gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac
     1381 tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa
     1441 atgagctgtt gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca
     1501 atttcacaca ggaaacagcc agtccgttta ggtgttttca cgagcacttc accaacaagg
     1561 accatagatt atgaaaatcg aagaacacca tcaccatcac cattccagcg gtaagcttat
     1621 gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt
     1681 ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg
     1741 gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg
     1801 tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat
     1861 gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga
     1921 tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga
     1981 ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac
     2041 atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt
     2101 tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa
     2161 acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg
     2221 gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa aatcggatct
     2281 ggaagttctg ttccaggggc ccctgtccag cggtctggtt ccgcgtggtt ccggtaccgc
     2341 ggcccagccg gccatggccc agggcgcgct ggtttgtgca gaagcactgt gggatcatgt
     2401 caccatggat gatcaggaac tgggtttcaa agccggtgat gtcattgaag ttatggatgc
     2461 caccaatcgt gaatggtggt ggggtcgtgt tgcagatggt gaaggttggt ttccagcctc
     2521 ttttgttcgt atgcgtgttg cggccgcata ataaaccgat acaattaaag gctccttttg
     2581 gagccttttt tttggagatt ttcaacgtga aaaaattatt attcgcaatt cctttagttg
     2641 ttcctttcta ttctcactcc gctgaaactg ttgaaagttg tttagcaaaa ccccatacag
     2701 aaaattcatt tactaacgtc tggaaagacg acaaaacttt agatcgttac gctaactatg
     2761 agggctgtct gtggaatgct acaggcgttg tagtttgtac tggtgacgaa actcagtgtc
     2821 tagctagagt ggcggtggct ctggttccgg tgattttgat tatgaaaaga tggcaaacgc
     2881 taataagggg gctatgaccg aaaatgccga tgaaaacgcg ctacagtctg acgctaaagg
     2941 caaacttgat tctgtcgcta ctgattacgg tgctgctatc gatggtttca ttggtgacgt
     3001 ttccggcctt gctaatggta atggtgctac tggtgatttt gctggctcta attcccaaat
     3061 ggctcaagtc ggtgacggtg ataattcacc tttaatgaat aatttccgtc aatatttacc
     3121 ttccctccct caatcggttg aatgtcgccc ttttgtcttt ggcgctggta aaccatatga
     3181 attttctatt gattgtgaca aaataaactt attccgtggt gtctttgcgt ttcttttata
     3241 tgttgccacc tttatgtatg tattttctac gtttgctaac atactgcgta ataaggagtc
     3301 ttaatcatgc cagttctttt ggctagcgcc gccctatacc ttgtctgcct ccccgcgttg
     3361 cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta
     3421 acggattcac cactccaaga attggagcca atcaattctt gcggagaact gtgaatgcgc
     3481 aaaccaaccc ttggcagaac atatccatcg cgtccgccat ctccagcagc cgcacgcggc
     3541 gcatctcggg cagcgttggg tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga
     3601 ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg
     3661 agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg
     3721 tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta
     3781 tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat
     3841 taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg
     3901 ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta
     3961 tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt
     4021 acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca
     4081 gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag
     4141 acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcaggatc cgcgaaattg
     4201 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     4261 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     4321 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     4381 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     4441 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     4501 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     4561 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     4621 ccgcgcttaa tgcgccgcta cagggcgcgt cgcggatcct gcctcgcgcg tttcggtgat
     4681 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     4741 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     4801 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat
     4861 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
     4921 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4981 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5041 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5101 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5161 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccagggtt
     5221 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5281 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5341 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5401 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5461 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5521 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     5581 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5641 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5701 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5761 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5821 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5881 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5941 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6001 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6061 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6121 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6181 caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6241 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatat tgtgcaaaaa
     6301 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6361 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6421 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6481 ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa ctttaaaagt
     6541 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6601 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6661 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6721 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6781 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6841 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     6901 gacattaacc ta
//
