LOCUS       sequence-191075-        6951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            PRAM1-1/1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6951)
  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 6951)
  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_PRAM1-1/1
FEATURES             Location/Qualifiers
     source          1..6951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     33..51
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        74..151
                     /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             152..1234
                     /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(171..190)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1477..1482,1483..1498,1499..1505)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1513..1529
                     /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     1518..1540
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1614..1631
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1647..2300
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2258..2280
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2307..2330
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2343..2360
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2622..2669
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4262..4717)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4399..4420)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4611..4630
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4759..4781)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4867..5007
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4922..4941
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5022..5039)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5193..5780)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5951..6742,6743..6811))
                     /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     6574..6593
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6812..6916)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
       61 ttcaagaatt cccgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg
      121 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
      181 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
      241 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
      301 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
      361 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
      421 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
      481 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
      541 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
      601 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
      661 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
      721 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
      781 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
      841 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
      901 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt
      961 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
     1021 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
     1081 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     1141 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     1201 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc
     1261 tcactcatta ggcacaattc tcatgtttga cagcttatca tcgactgcac ggtgcaccaa
     1321 tgcttctggc gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac
     1381 tgcataattc gtgtcgctca aggcgcactc ccgttctgga taatgttttt tgcgccgaca
     1441 tcataacggt tctggcaaat attctgaaat gagctgttga caattaatca tcggctcgta
     1501 taatgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagccag tccgtttagg
     1561 tgttttcacg agcacttcac caacaaggac catagattat gaaaatcgaa gaacaccatc
     1621 accatcacca ttccagcggt aagcttatgt cccctatact aggttattgg aaaattaagg
     1681 gccttgtgca acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt
     1741 tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt
     1801 ttcccaatct tccttattat attgatggtg atgttaaatt aacacagtct atggccatca
     1861 tacgttatat agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga
     1921 tttcaatgct tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata
     1981 gtaaagactt tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa
     2041 tgttcgaaga tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg
     2101 acttcatgtt gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg
     2161 cgttcccaaa attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt
     2221 acttgaaatc cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg
     2281 gtggcgacca tcctccaaaa tcggatctgg aagttctgtt ccaggggccc ctgtccagcg
     2341 gtctggttcc gcgtggttcc ggtaccgcgg cccagccggc ccgtgaattc cgtaaaaaat
     2401 tcaaattcga aggtgaaatc gttgtccata ccaaaatgat gattgacccg aacgccaaaa
     2461 cccgtcgtgg cggtggcaaa catctgggta ttcgtcgcgg cgaaattctg gaagtgatcg
     2521 aatttaccag caacgaagaa atgctgtgcc gtgatccgaa gggtaaatat ggctacgttc
     2581 cgcgcacggc gctgctgccg ctggaagcgg ccgcataata aaccgataca attaaaggct
     2641 ccttttggag cctttttttt ggagattttc aacgtgaaaa aattattatt cgcaattcct
     2701 ttagttgttc ctttctattc tcactccgct gaaactgttg aaagttgttt agcaaaaccc
     2761 catacagaaa attcatttac taacgtctgg aaagacgaca aaactttaga tcgttacgct
     2821 aactatgagg gctgtctgtg gaatgctaca ggcgttgtag tttgtactgg tgacgaaact
     2881 cagtgtctag ctagagtggc ggtggctctg gttccggtga ttttgattat gaaaagatgg
     2941 caaacgctaa taagggggct atgaccgaaa atgccgatga aaacgcgcta cagtctgacg
     3001 ctaaaggcaa acttgattct gtcgctactg attacggtgc tgctatcgat ggtttcattg
     3061 gtgacgtttc cggccttgct aatggtaatg gtgctactgg tgattttgct ggctctaatt
     3121 cccaaatggc tcaagtcggt gacggtgata attcaccttt aatgaataat ttccgtcaat
     3181 atttaccttc cctccctcaa tcggttgaat gtcgcccttt tgtctttggc gctggtaaac
     3241 catatgaatt ttctattgat tgtgacaaaa taaacttatt ccgtggtgtc tttgcgtttc
     3301 ttttatatgt tgccaccttt atgtatgtat tttctacgtt tgctaacata ctgcgtaata
     3361 aggagtctta atcatgccag ttcttttggc tagcgccgcc ctataccttg tctgcctccc
     3421 cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc tgaatggaag ccggcggcac
     3481 ctcgctaacg gattcaccac tccaagaatt ggagccaatc aattcttgcg gagaactgtg
     3541 aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt ccgccatctc cagcagccgc
     3601 acgcggcgca tctcgggcag cgttgggtcc tggccacggg tgcgcatgat cgtgctcctg
     3661 tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg
     3721 atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca
     3781 tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc
     3841 accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca
     3901 tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc
     3961 cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta
     4021 acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat
     4081 cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc
     4141 tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa
     4201 caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg caggatccgc
     4261 gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     4321 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     4381 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     4441 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     4501 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     4561 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     4621 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     4681 acacccgccg cgcttaatgc gccgctacag ggcgcgtcgc ggatcctgcc tcgcgcgttt
     4741 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     4801 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     4861 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     4921 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga
     4981 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     5041 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5101 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5161 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5221 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5281 cagggtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5341 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5401 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5461 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5521 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5581 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     5641 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5701 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5761 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5821 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5881 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5941 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6001 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6061 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6121 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6181 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6241 gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta
     6301 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6361 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6421 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6481 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6541 gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt
     6601 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6661 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6721 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6781 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6841 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6901 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa g
//
