LOCUS       sequence-191907-        6957 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            KALRN-3/4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6957)
  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 6957)
  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_KALRN-3/4
FEATURES             Location/Qualifiers
     source          1..6957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     36..54
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        77..154
                     /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             155..1237
                     /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(174..193)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1480..1485,1486..1501,1502..1508)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1516..1532
                     /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     1521..1543
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1617..1634
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1650..2303
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2261..2283
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2310..2333
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2346..2363
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2631..2678
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4271..4726)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4408..4429)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4620..4639
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4768..4790)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4876..5016
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4931..4950
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5031..5048)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5202..5789)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5960..6751,6752..6820))
                     /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     6583..6602
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6821..6925)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
       61 gtcttcaaga attcccgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata
      121 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
      181 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
      241 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
      301 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
      361 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
      421 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
      481 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
      541 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
      601 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
      661 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
      721 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
      781 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
      841 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
      901 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
      961 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     1021 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     1081 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     1141 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     1201 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     1261 agctcactca ttaggcacaa ttctcatgtt tgacagctta tcatcgactg cacggtgcac
     1321 caatgcttct ggcgtcaggc agccatcgga agctgtggta tggctgtgca ggtcgtaaat
     1381 cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg
     1441 acatcataac ggttctggca aatattctga aatgagctgt tgacaattaa tcatcggctc
     1501 gtataatgtg tggaattgtg agcggataac aatttcacac aggaaacagc cagtccgttt
     1561 aggtgttttc acgagcactt caccaacaag gaccatagat tatgaaaatc gaagaacacc
     1621 atcaccatca ccattccagc ggtaagctta tgtcccctat actaggttat tggaaaatta
     1681 agggccttgt gcaacccact cgacttcttt tggaatatct tgaagaaaaa tatgaagagc
     1741 atttgtatga gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg
     1801 agtttcccaa tcttccttat tatattgatg gtgatgttaa attaacacag tctatggcca
     1861 tcatacgtta tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag
     1921 agatttcaat gcttgaagga gcggttttgg atattagata cggtgtttcg agaattgcat
     1981 atagtaaaga ctttgaaact ctcaaagttg attttcttag caagctacct gaaatgctga
     2041 aaatgttcga agatcgttta tgtcataaaa catatttaaa tggtgatcat gtaacccatc
     2101 ctgacttcat gttgtatgac gctcttgatg ttgttttata catggaccca atgtgcctgg
     2161 atgcgttccc aaaattagtt tgttttaaaa aacgtattga agctatccca caaattgata
     2221 agtacttgaa atccagcaag tatatagcat ggcctttgca gggctggcaa gccacgtttg
     2281 gtggtggcga ccatcctcca aaatcggatc tggaagttct gttccagggg cccctgtcca
     2341 gcggtctggt tccgcgtggt tccggtaccg cggcccagcc ggccatggcc cagggcgcgc
     2401 tgagttctat ggccgtcatt aaagattact atgccctgaa agaaaacgaa atctgtgtct
     2461 cccagggtga agttgttcaa gttctggccg ttaaccagca gaatatgtgc ctggtttatc
     2521 aaccagcatc cgatcattct ccagcagcag aaggttgggt tccaggttct attctggcac
     2581 cactgaccaa agcaaccgca gcagaatctt ctgatgcggc cgcataataa accgatacaa
     2641 ttaaaggctc cttttggagc cttttttttg gagattttca acgtgaaaaa attattattc
     2701 gcaattcctt tagttgttcc tttctattct cactccgctg aaactgttga aagttgttta
     2761 gcaaaacccc atacagaaaa ttcatttact aacgtctgga aagacgacaa aactttagat
     2821 cgttacgcta actatgaggg ctgtctgtgg aatgctacag gcgttgtagt ttgtactggt
     2881 gacgaaactc agtgtctagc tagagtggcg gtggctctgg ttccggtgat tttgattatg
     2941 aaaagatggc aaacgctaat aagggggcta tgaccgaaaa tgccgatgaa aacgcgctac
     3001 agtctgacgc taaaggcaaa cttgattctg tcgctactga ttacggtgct gctatcgatg
     3061 gtttcattgg tgacgtttcc ggccttgcta atggtaatgg tgctactggt gattttgctg
     3121 gctctaattc ccaaatggct caagtcggtg acggtgataa ttcaccttta atgaataatt
     3181 tccgtcaata tttaccttcc ctccctcaat cggttgaatg tcgccctttt gtctttggcg
     3241 ctggtaaacc atatgaattt tctattgatt gtgacaaaat aaacttattc cgtggtgtct
     3301 ttgcgtttct tttatatgtt gccaccttta tgtatgtatt ttctacgttt gctaacatac
     3361 tgcgtaataa ggagtcttaa tcatgccagt tcttttggct agcgccgccc tataccttgt
     3421 ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct gaatggaagc
     3481 cggcggcacc tcgctaacgg attcaccact ccaagaattg gagccaatca attcttgcgg
     3541 agaactgtga atgcgcaaac caacccttgg cagaacatat ccatcgcgtc cgccatctcc
     3601 agcagccgca cgcggcgcat ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc
     3661 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
     3721 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
     3781 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     3841 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     3901 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     3961 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     4021 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     4081 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     4141 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     4201 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     4261 aggatccgcg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     4321 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     4381 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     4441 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     4501 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     4561 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     4621 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     4681 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcgcg gatcctgcct
     4741 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     4801 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     4861 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     4921 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
     4981 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     5041 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5101 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5161 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5221 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5281 taaagatacc agggtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5341 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5401 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5461 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5521 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5581 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5641 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5701 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5761 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5821 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5881 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5941 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6001 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6061 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6121 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6181 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6241 cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt
     6301 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6361 ccatattgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6421 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6481 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6541 gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata
     6601 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6661 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6721 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6781 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6841 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6901 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtct
//
