LOCUS       sequence-191106-        6900 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            KALRN-1/4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6900)
  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 6900)
  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-1/4
FEATURES             Location/Qualifiers
     source          1..6900
                     /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      2547..2594
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4187..4642)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4324..4345)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4536..4555
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4684..4706)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4792..4932
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4847..4866
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4947..4964)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5118..5705)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5876..6667,6668..6736))
                     /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     6499..6518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6737..6841)
                     /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 gccggccgaa ctgaccgtcg tcctgcaaga tttttctgcg ggccattcaa
     2401 gcgaactgac catccaagtc ggccaaaccg tcgaactgct ggaacgtccg agtgaacgtc
     2461 cgggttggtg cctggtgcgt accacggaac gtagcccgcc gctggaaggt ctggttccga
     2521 gctctgcgct ggcggccgca taataaaccg atacaattaa aggctccttt tggagccttt
     2581 tttttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc
     2641 tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aaccccatac agaaaattca
     2701 tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta tgagggctgt
     2761 ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg aaactcagtg tctagctaga
     2821 gtggcggtgg ctctggttcc ggtgattttg attatgaaaa gatggcaaac gctaataagg
     2881 gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc tgacgctaaa ggcaaacttg
     2941 attctgtcgc tactgattac ggtgctgcta tcgatggttt cattggtgac gtttccggcc
     3001 ttgctaatgg taatggtgct actggtgatt ttgctggctc taattcccaa atggctcaag
     3061 tcggtgacgg tgataattca cctttaatga ataatttccg tcaatattta ccttccctcc
     3121 ctcaatcggt tgaatgtcgc ccttttgtct ttggcgctgg taaaccatat gaattttcta
     3181 ttgattgtga caaaataaac ttattccgtg gtgtctttgc gtttctttta tatgttgcca
     3241 cctttatgta tgtattttct acgtttgcta acatactgcg taataaggag tcttaatcat
     3301 gccagttctt ttggctagcg ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg
     3361 tgcatggagc cgggccacct cgacctgaat ggaagccggc ggcacctcgc taacggattc
     3421 accactccaa gaattggagc caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac
     3481 ccttggcaga acatatccat cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg
     3541 ggcagcgttg ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg
     3601 ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg
     3661 tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt
     3721 ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg
     3781 atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag
     3841 cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt
     3901 ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc
     3961 atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag
     4021 gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag
     4081 acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt
     4141 gaatcgcttc acgaccacgc tgatgagctt taccgcagga tccgcgaaat tgtaagcgtt
     4201 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     4261 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     4321 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     4381 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     4441 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     4501 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     4561 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     4621 aatgcgccgc tacagggcgc gtcgcggatc ctgcctcgcg cgtttcggtg atgacggtga
     4681 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     4741 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     4801 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     4861 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     4921 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4981 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5041 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5101 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5161 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggg tttccccctg
     5221 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5281 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5341 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5401 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5461 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5521 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     5581 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5641 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5701 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5761 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5821 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5881 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5941 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6001 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6061 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6121 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6181 ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6241 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6301 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6361 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6421 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6481 gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6541 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6601 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6661 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6721 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6781 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6841 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
//
