LOCUS       sequence-191896-        6930 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            KALRN-2/4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6930)
  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 6930)
  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-2/4
FEATURES             Location/Qualifiers
     source          1..6930
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..45
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        68..145
                     /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             146..1228
                     /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(165..184)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1471..1476,1477..1492,1493..1499)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1507..1523
                     /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     1512..1534
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1608..1625
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1641..2294
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2252..2274
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2301..2324
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2337..2354
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2595..2642
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4235..4690)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4372..4393)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4584..4603
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4732..4754)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4840..4980
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4895..4914
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4995..5012)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5166..5753)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5924..6715,6716..6784))
                     /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     6547..6566
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6785..6889)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcaag
       61 aattcccgac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga
      121 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
      181 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
      241 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
      301 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
      361 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
      421 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
      481 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
      541 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
      601 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
      661 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
      721 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
      781 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
      841 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
      901 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
      961 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     1021 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     1081 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     1141 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     1201 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     1261 attaggcaca attctcatgt ttgacagctt atcatcgact gcacggtgca ccaatgcttc
     1321 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata
     1381 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa
     1441 cggttctggc aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt
     1501 gtggaattgt gagcggataa caatttcaca caggaaacag ccagtccgtt taggtgtttt
     1561 cacgagcact tcaccaacaa ggaccataga ttatgaaaat cgaagaacac catcaccatc
     1621 accattccag cggtaagctt atgtccccta tactaggtta ttggaaaatt aagggccttg
     1681 tgcaacccac tcgacttctt ttggaatatc ttgaagaaaa atatgaagag catttgtatg
     1741 agcgcgatga aggtgataaa tggcgaaaca aaaagtttga attgggtttg gagtttccca
     1801 atcttcctta ttatattgat ggtgatgtta aattaacaca gtctatggcc atcatacgtt
     1861 atatagctga caagcacaac atgttgggtg gttgtccaaa agagcgtgca gagatttcaa
     1921 tgcttgaagg agcggttttg gatattagat acggtgtttc gagaattgca tatagtaaag
     1981 actttgaaac tctcaaagtt gattttctta gcaagctacc tgaaatgctg aaaatgttcg
     2041 aagatcgttt atgtcataaa acatatttaa atggtgatca tgtaacccat cctgacttca
     2101 tgttgtatga cgctcttgat gttgttttat acatggaccc aatgtgcctg gatgcgttcc
     2161 caaaattagt ttgttttaaa aaacgtattg aagctatccc acaaattgat aagtacttga
     2221 aatccagcaa gtatatagca tggcctttgc agggctggca agccacgttt ggtggtggcg
     2281 accatcctcc aaaatcggat ctggaagttc tgttccaggg gcccctgtcc agcggtctgg
     2341 ttccgcgtgg ttccggtacc gcggcccagc cggccatggc ccagggcgcg ctggaactga
     2401 ccgttgtcct gcaagatttc tctgccggtc attcttctga actgaccatt caagttggtc
     2461 agaccgttga actgctggaa cgtccatctg aacgtcctgg ttggtgttta gttcgtacca
     2521 ccgaacgttc tccaccactg gaaggtctgg ttccatcttc tgccctgtgt atttcccatg
     2581 cggccgcata ataaaccgat acaattaaag gctccttttg gagccttttt tttggagatt
     2641 ttcaacgtga aaaaattatt attcgcaatt cctttagttg ttcctttcta ttctcactcc
     2701 gctgaaactg ttgaaagttg tttagcaaaa ccccatacag aaaattcatt tactaacgtc
     2761 tggaaagacg acaaaacttt agatcgttac gctaactatg agggctgtct gtggaatgct
     2821 acaggcgttg tagtttgtac tggtgacgaa actcagtgtc tagctagagt ggcggtggct
     2881 ctggttccgg tgattttgat tatgaaaaga tggcaaacgc taataagggg gctatgaccg
     2941 aaaatgccga tgaaaacgcg ctacagtctg acgctaaagg caaacttgat tctgtcgcta
     3001 ctgattacgg tgctgctatc gatggtttca ttggtgacgt ttccggcctt gctaatggta
     3061 atggtgctac tggtgatttt gctggctcta attcccaaat ggctcaagtc ggtgacggtg
     3121 ataattcacc tttaatgaat aatttccgtc aatatttacc ttccctccct caatcggttg
     3181 aatgtcgccc ttttgtcttt ggcgctggta aaccatatga attttctatt gattgtgaca
     3241 aaataaactt attccgtggt gtctttgcgt ttcttttata tgttgccacc tttatgtatg
     3301 tattttctac gtttgctaac atactgcgta ataaggagtc ttaatcatgc cagttctttt
     3361 ggctagcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg
     3421 ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga
     3481 attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac
     3541 atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg
     3601 tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg
     3661 ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc
     3721 tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg
     3781 taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca
     3841 ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga
     3901 ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa
     3961 cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt
     4021 ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc
     4081 aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt
     4141 ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac
     4201 gaccacgctg atgagcttta ccgcaggatc cgcgaaattg taagcgttaa tattttgtta
     4261 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     4321 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     4381 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     4441 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     4501 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     4561 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     4621 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     4681 cagggcgcgt cgcggatcct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     4741 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     4801 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     4861 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     4921 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     4981 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5041 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5101 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5161 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5221 aggtggcgaa acccgacagg actataaaga taccagggtt tccccctgga agctccctcg
     5281 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5341 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5401 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5461 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5521 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5581 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     5641 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5701 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5761 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5821 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5881 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5941 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6001 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6061 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6121 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6181 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg
     6241 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6301 gatcaaggcg agttacatga tcccccatat tgtgcaaaaa agcggttagc tccttcggtc
     6361 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6421 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6481 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6541 cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6601 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6661 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6721 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6781 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6841 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6901 gaaaagtgcc acctgacgtc taagaaacca
//
