LOCUS       sequence-192351-        6909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            GRAP2-1/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6909)
  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 6909)
  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_GRAP2-1/2
FEATURES             Location/Qualifiers
     source          1..6909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..26
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        49..126
                     /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             127..1209
                     /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(146..165)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1452..1457,1458..1473,1474..1480)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1488..1504
                     /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     1493..1515
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1589..1606
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1622..2275
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2233..2255
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2282..2305
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2318..2335
                     /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 ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaattcccga caccatcgaa
       61 tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
      121 gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag
      181 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
      241 gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
      301 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
      361 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
      421 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
      481 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
      541 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
      601 attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
      661 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
      721 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
      781 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
      841 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
      901 gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac
      961 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
     1021 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
     1081 ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct
     1141 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     1201 gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac aattctcatg
     1261 tttgacagct tatcatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg
     1321 gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg
     1381 cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct
     1441 gaaatgagct gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata
     1501 acaatttcac acaggaaaca gccagtccgt ttaggtgttt tcacgagcac ttcaccaaca
     1561 aggaccatag attatgaaaa tcgaagaaca ccatcaccat caccattcca gcggtaagct
     1621 tatgtcccct atactaggtt attggaaaat taagggcctt gtgcaaccca ctcgacttct
     1681 tttggaatat cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg aaggtgataa
     1741 atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttcctt attatattga
     1801 tggtgatgtt aaattaacac agtctatggc catcatacgt tatatagctg acaagcacaa
     1861 catgttgggt ggttgtccaa aagagcgtgc agagatttca atgcttgaag gagcggtttt
     1921 ggatattaga tacggtgttt cgagaattgc atatagtaaa gactttgaaa ctctcaaagt
     1981 tgattttctt agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa
     2041 aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatg acgctcttga
     2101 tgttgtttta tacatggacc caatgtgcct ggatgcgttc ccaaaattag tttgttttaa
     2161 aaaacgtatt gaagctatcc cacaaattga taagtacttg aaatccagca agtatatagc
     2221 atggcctttg cagggctggc aagccacgtt tggtggtggc gaccatcctc caaaatcgga
     2281 tctggaagtt ctgttccagg ggcccctgtc cagcggtctg gttccgcgtg gttccggtac
     2341 cgcggcccag ccggccatgg cccagggcgc gctgatggaa gccgtcgcca aatttgattt
     2401 caccgcctcc ggtgaagatg aactgtcctt tcataccggt gatgttctga aaatcctgtc
     2461 caaccaggaa gaatggttca aagcagaact gggttcccag gaaggttatg ttccgaaaaa
     2521 cttcattgat attcagttcg 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 gacattaac
//
