LOCUS       sequence-192233-        6912 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            GRAP2-2/2.
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 Apr 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_GRAP2-2/2
FEATURES             Location/Qualifiers
     source          1..6912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..20
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        43..120
                     /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             121..1203
                     /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(140..159)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1446..1451,1452..1467,1468..1474)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1482..1498
                     /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     1487..1509
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1583..1600
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1616..2269
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2227..2249
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2276..2299
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2312..2329
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2552..2599
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4192..4647)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4329..4350)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4541..4560
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4689..4711)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4797..4937
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4852..4871
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4952..4969)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5123..5710)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5881..6672,6673..6741))
                     /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     6504..6523
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6742..6846)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaataggcgt atcacgaggc cctttcgtct tcaagaattc ccgacaccat cgaatggtgc
       61 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
      121 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
      181 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
      241 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
      301 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
      361 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
      421 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
      481 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
      541 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
      601 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
      661 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
      721 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
      781 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
      841 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
      901 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
      961 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     1021 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     1081 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc
     1141 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     1201 tgagcgcaac gcaattaatg taagttagct cactcattag gcacaattct catgtttgac
     1261 agcttatcat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc atcggaagct
     1321 gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc
     1381 cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata ttctgaaatg
     1441 agctgttgac aattaatcat cggctcgtat aatgtgtgga attgtgagcg gataacaatt
     1501 tcacacagga aacagccagt ccgtttaggt gttttcacga gcacttcacc aacaaggacc
     1561 atagattatg aaaatcgaag aacaccatca ccatcaccat tccagcggta agcttatgtc
     1621 ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga
     1681 atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg
     1741 aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga
     1801 tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt
     1861 gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat
     1921 tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt
     1981 tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata
     2041 tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt
     2101 tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg
     2161 tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc
     2221 tttgcagggc tggcaagcca cgtttggtgg tggcgaccat cctccaaaat cggatctgga
     2281 agttctgttc caggggcccc tgtccagcgg tctggttccg cgtggttccg gtaccgcggc
     2341 ccagccggcc atggcccagg gcgcgctgcg ttgggcacgt gcactgtatg atttcgaagc
     2401 cctggaagat gatgaactgg gttttcattc cggtgaagtc gttgaagttc tggattcctc
     2461 caatccatct tggtggaccg gtcgtctgca taacaaactc ggtctctttc cggcaaacta
     2521 tgttgcacca atgaccgcgg ccgcataata aaccgataca attaaaggct ccttttggag
     2581 cctttttttt ggagattttc aacgtgaaaa aattattatt cgcaattcct ttagttgttc
     2641 ctttctattc tcactccgct gaaactgttg aaagttgttt agcaaaaccc catacagaaa
     2701 attcatttac taacgtctgg aaagacgaca aaactttaga tcgttacgct aactatgagg
     2761 gctgtctgtg gaatgctaca ggcgttgtag tttgtactgg tgacgaaact cagtgtctag
     2821 ctagagtggc ggtggctctg gttccggtga ttttgattat gaaaagatgg caaacgctaa
     2881 taagggggct atgaccgaaa atgccgatga aaacgcgcta cagtctgacg ctaaaggcaa
     2941 acttgattct gtcgctactg attacggtgc tgctatcgat ggtttcattg gtgacgtttc
     3001 cggccttgct aatggtaatg gtgctactgg tgattttgct ggctctaatt cccaaatggc
     3061 tcaagtcggt gacggtgata attcaccttt aatgaataat ttccgtcaat atttaccttc
     3121 cctccctcaa tcggttgaat gtcgcccttt tgtctttggc gctggtaaac catatgaatt
     3181 ttctattgat tgtgacaaaa taaacttatt ccgtggtgtc tttgcgtttc ttttatatgt
     3241 tgccaccttt atgtatgtat tttctacgtt tgctaacata ctgcgtaata aggagtctta
     3301 atcatgccag ttcttttggc tagcgccgcc ctataccttg tctgcctccc cgcgttgcgt
     3361 cgcggtgcat ggagccgggc cacctcgacc tgaatggaag ccggcggcac ctcgctaacg
     3421 gattcaccac tccaagaatt ggagccaatc aattcttgcg gagaactgtg aatgcgcaaa
     3481 ccaacccttg gcagaacata tccatcgcgt ccgccatctc cagcagccgc acgcggcgca
     3541 tctcgggcag cgttgggtcc tggccacggg tgcgcatgat cgtgctcctg tcgttgagga
     3601 cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc
     3661 gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct
     3721 tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt
     3781 tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa
     3841 cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca
     3901 gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg
     3961 tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca
     4021 cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa
     4081 gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca
     4141 tctgtgaatc gcttcacgac cacgctgatg agctttaccg caggatccgc gaaattgtaa
     4201 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4261 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4321 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4381 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     4441 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4501 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4561 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     4621 cgcttaatgc gccgctacag ggcgcgtcgc ggatcctgcc tcgcgcgttt cggtgatgac
     4681 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     4741 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
     4801 gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag
     4861 agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga
     4921 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4981 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5041 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5101 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5161 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac cagggtttcc
     5221 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5281 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5341 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5401 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5461 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5521 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     5581 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5641 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5701 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5761 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5821 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5881 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5941 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6001 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6061 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6121 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6181 cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6241 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatattgt gcaaaaaagc
     6301 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6361 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6421 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6481 ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct
     6541 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6601 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6661 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6721 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6781 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6841 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     6901 attaacctat aa
//
