LOCUS       sequence-192604-        6894 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            SH3RF1-1/4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6894)
  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 6894)
  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_SH3RF1-1/4
FEATURES             Location/Qualifiers
     source          1..6894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..27
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        50..127
                     /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             128..1210
                     /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(147..166)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1453..1458,1459..1474,1475..1481)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1489..1505
                     /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     1494..1516
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1590..1607
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1623..2276
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2234..2256
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2283..2306
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2319..2336
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2541..2588
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4181..4636)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4318..4339)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4530..4549
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4678..4700)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4786..4926
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4841..4860
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4941..4958)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5112..5699)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5870..6661,6662..6730))
                     /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     6493..6512
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6731..6835)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattcccg acaccatcga
       61 atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
      121 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
      181 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
      241 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
      301 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
      361 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
      421 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
      481 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
      541 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
      601 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
      661 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
      721 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
      781 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
      841 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
      901 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
      961 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     1021 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     1081 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
     1141 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     1201 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca caattctcat
     1261 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
     1321 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
     1381 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
     1441 tgaaatgagc tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat
     1501 aacaatttca cacaggaaac agccagtccg tttaggtgtt ttcacgagca cttcaccaac
     1561 aaggaccata gattatgaaa atcgaagaac accatcacca tcaccattcc agcggtaagc
     1621 ttatgtcccc tatactaggt tattggaaaa ttaagggcct tgtgcaaccc actcgacttc
     1681 ttttggaata tcttgaagaa aaatatgaag agcatttgta tgagcgcgat gaaggtgata
     1741 aatggcgaaa caaaaagttt gaattgggtt tggagtttcc caatcttcct tattatattg
     1801 atggtgatgt taaattaaca cagtctatgg ccatcatacg ttatatagct gacaagcaca
     1861 acatgttggg tggttgtcca aaagagcgtg cagagatttc aatgcttgaa ggagcggttt
     1921 tggatattag atacggtgtt tcgagaattg catatagtaa agactttgaa actctcaaag
     1981 ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt ttatgtcata
     2041 aaacatattt aaatggtgat catgtaaccc atcctgactt catgttgtat gacgctcttg
     2101 atgttgtttt atacatggac ccaatgtgcc tggatgcgtt cccaaaatta gtttgtttta
     2161 aaaaacgtat tgaagctatc ccacaaattg ataagtactt gaaatccagc aagtatatag
     2221 catggccttt gcagggctgg caagccacgt ttggtggtgg cgaccatcct ccaaaatcgg
     2281 atctggaagt tctgttccag gggcccctgt ccagcggtct ggttccgcgt ggttccggta
     2341 ccgcggccca gccggcccca tgcgcaaaag ccctgtacaa ctatgaaggt aaagaaccgg
     2401 gtgatctgaa attctccaaa ggtgacatta tcattctgcg tcgtcaagtc gatgaaaact
     2461 ggtatcatgg tgaagtcaac ggtattcatg gtttctttcc gaccaacttt gttcagatta
     2521 tcaaagcggc cgcataataa accgatacaa ttaaaggctc cttttggagc cttttttttg
     2581 gagattttca acgtgaaaaa attattattc gcaattcctt tagttgttcc tttctattct
     2641 cactccgctg aaactgttga aagttgttta gcaaaacccc atacagaaaa ttcatttact
     2701 aacgtctgga aagacgacaa aactttagat cgttacgcta actatgaggg ctgtctgtgg
     2761 aatgctacag gcgttgtagt ttgtactggt gacgaaactc agtgtctagc tagagtggcg
     2821 gtggctctgg ttccggtgat tttgattatg aaaagatggc aaacgctaat aagggggcta
     2881 tgaccgaaaa tgccgatgaa aacgcgctac agtctgacgc taaaggcaaa cttgattctg
     2941 tcgctactga ttacggtgct gctatcgatg gtttcattgg tgacgtttcc ggccttgcta
     3001 atggtaatgg tgctactggt gattttgctg gctctaattc ccaaatggct caagtcggtg
     3061 acggtgataa ttcaccttta atgaataatt tccgtcaata tttaccttcc ctccctcaat
     3121 cggttgaatg tcgccctttt gtctttggcg ctggtaaacc atatgaattt tctattgatt
     3181 gtgacaaaat aaacttattc cgtggtgtct ttgcgtttct tttatatgtt gccaccttta
     3241 tgtatgtatt ttctacgttt gctaacatac tgcgtaataa ggagtcttaa tcatgccagt
     3301 tcttttggct agcgccgccc tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg
     3361 gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg attcaccact
     3421 ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac caacccttgg
     3481 cagaacatat ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat ctcgggcagc
     3541 gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc
     3601 tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc
     3661 gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt
     3721 gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc
     3781 atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg
     3841 cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc
     3901 tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc
     3961 tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca
     4021 gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta
     4081 acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg
     4141 cttcacgacc acgctgatga gctttaccgc aggatccgcg aaattgtaag cgttaatatt
     4201 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     4261 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     4321 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     4381 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     4441 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     4501 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     4561 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     4621 ccgctacagg gcgcgtcgcg gatcctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     4681 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     4741 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     4801 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta
     4861 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     4921 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4981 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5041 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5101 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5161 agtcagaggt ggcgaaaccc gacaggacta taaagatacc agggtttccc cctggaagct
     5221 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5281 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5341 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5401 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5461 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5521 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     5581 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5641 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5701 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5761 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5821 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5881 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5941 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6001 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6061 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6121 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6181 ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6241 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6301 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6361 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6421 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6481 cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6541 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6601 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6661 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6721 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6781 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6841 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaa
//
