LOCUS       sequence-191016-        6915 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            CASKIN2-1/1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6915)
  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 6915)
  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_CASKIN2-1/1
FEATURES             Location/Qualifiers
     source          1..6915
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        39..116
                     /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             117..1199
                     /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(136..155)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1442..1447,1448..1463,1464..1470)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1478..1494
                     /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     1483..1505
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1579..1596
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1612..2265
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2223..2245
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2272..2295
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2308..2325
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2551..2598
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4191..4646)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4328..4349)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4540..4559
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4688..4710)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4796..4936
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4851..4870
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4951..4968)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5122..5709)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5880..6671,6672..6740))
                     /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     6503..6522
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6741..6845)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(6913..6915,1..16)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aggcgtatca cgaggccctt tcgtcttcaa gaattcccga caccatcgaa tggtgcaaaa
       61 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
      121 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
      181 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
      241 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
      301 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
      361 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
      421 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
      481 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
      541 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
      601 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
      661 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
      721 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
      781 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
      841 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
      901 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
      961 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     1021 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     1081 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     1141 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     1201 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac aattctcatg tttgacagct
     1261 tatcatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
     1321 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
     1381 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
     1441 gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac
     1501 acaggaaaca gccagtccgt ttaggtgttt tcacgagcac ttcaccaaca aggaccatag
     1561 attatgaaaa tcgaagaaca ccatcaccat caccattcca gcggtaagct tatgtcccct
     1621 atactaggtt attggaaaat taagggcctt gtgcaaccca ctcgacttct tttggaatat
     1681 cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg aaggtgataa atggcgaaac
     1741 aaaaagtttg aattgggttt ggagtttccc aatcttcctt attatattga tggtgatgtt
     1801 aaattaacac agtctatggc catcatacgt tatatagctg acaagcacaa catgttgggt
     1861 ggttgtccaa aagagcgtgc agagatttca atgcttgaag gagcggtttt ggatattaga
     1921 tacggtgttt cgagaattgc atatagtaaa gactttgaaa ctctcaaagt tgattttctt
     1981 agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa aacatattta
     2041 aatggtgatc atgtaaccca tcctgacttc atgttgtatg acgctcttga tgttgtttta
     2101 tacatggacc caatgtgcct ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt
     2161 gaagctatcc cacaaattga taagtacttg aaatccagca agtatatagc atggcctttg
     2221 cagggctggc aagccacgtt tggtggtggc gaccatcctc caaaatcgga tctggaagtt
     2281 ctgttccagg ggcccctgtc cagcggtctg gttccgcgtg gttccggtac cgcggcccag
     2341 ccggccctga aagttcgtgc tctgaaagac ttctggaatc tgcatgaccc gacggctctg
     2401 aatgttcgtg ctggcgatgt tattacggtt ctggaacagc atccggatgg ccgttggaaa
     2461 ggtcatattc acgaaagcca acgtggcacc gaccgcattg gttattttcc gccgggcatc
     2521 gtcgaagtgg tttctgcggc cgcataataa accgatacaa ttaaaggctc cttttggagc
     2581 cttttttttg gagattttca acgtgaaaaa attattattc gcaattcctt tagttgttcc
     2641 tttctattct cactccgctg aaactgttga aagttgttta gcaaaacccc atacagaaaa
     2701 ttcatttact aacgtctgga aagacgacaa aactttagat cgttacgcta actatgaggg
     2761 ctgtctgtgg aatgctacag gcgttgtagt ttgtactggt gacgaaactc agtgtctagc
     2821 tagagtggcg gtggctctgg ttccggtgat tttgattatg aaaagatggc aaacgctaat
     2881 aagggggcta tgaccgaaaa tgccgatgaa aacgcgctac agtctgacgc taaaggcaaa
     2941 cttgattctg tcgctactga ttacggtgct gctatcgatg gtttcattgg tgacgtttcc
     3001 ggccttgcta atggtaatgg tgctactggt gattttgctg gctctaattc ccaaatggct
     3061 caagtcggtg acggtgataa ttcaccttta atgaataatt tccgtcaata tttaccttcc
     3121 ctccctcaat cggttgaatg tcgccctttt gtctttggcg ctggtaaacc atatgaattt
     3181 tctattgatt gtgacaaaat aaacttattc cgtggtgtct ttgcgtttct tttatatgtt
     3241 gccaccttta tgtatgtatt ttctacgttt gctaacatac tgcgtaataa ggagtcttaa
     3301 tcatgccagt tcttttggct agcgccgccc tataccttgt ctgcctcccc gcgttgcgtc
     3361 gcggtgcatg gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg
     3421 attcaccact ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac
     3481 caacccttgg cagaacatat ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat
     3541 ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac
     3601 ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg
     3661 aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt
     3721 cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt
     3781 ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac
     3841 gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag
     3901 ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt
     3961 gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac
     4021 ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag
     4081 ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat
     4141 ctgtgaatcg cttcacgacc acgctgatga gctttaccgc aggatccgcg aaattgtaag
     4201 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     4261 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     4321 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     4381 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     4441 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     4501 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     4561 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     4621 gcttaatgcg ccgctacagg gcgcgtcgcg gatcctgcct cgcgcgtttc ggtgatgacg
     4681 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     4741 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     4801 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     4861 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
     4921 aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4981 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5041 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5101 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5161 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc agggtttccc
     5221 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5281 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5341 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5401 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5461 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5521 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     5581 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5641 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5701 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5761 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5821 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5881 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5941 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6001 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6061 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6121 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6181 gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6241 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6301 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6361 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6421 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6481 tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6541 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6601 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6661 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6721 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6781 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6841 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     6901 ttaacctata aaaat
//
