LOCUS       sequence-191780-        6900 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            CACNB2-1/2.
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_CACNB2-1/2
FEATURES             Location/Qualifiers
     source          1..6900
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..29
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        52..129
                     /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             130..1212
                     /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(149..168)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1455..1460,1461..1476,1477..1483)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1491..1507
                     /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     1496..1518
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1592..1609
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1625..2278
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2236..2258
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2285..2308
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2321..2338
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2549..2596
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4189..4644)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4326..4347)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4538..4557
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4686..4708)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4794..4934
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4849..4868
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4949..4966)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5120..5707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5878..6669,6670..6738))
                     /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     6501..6520
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6739..6843)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattcc cgacaccatc
       61 gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
      121 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
      181 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
      241 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
      301 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
      361 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
      421 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
      481 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
      541 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
      601 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
      661 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
      721 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
      781 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
      841 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
      901 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
      961 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     1021 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     1081 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     1141 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     1201 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg cacaattctc
     1261 atgtttgaca gcttatcatc gactgcacgg tgcaccaatg cttctggcgt caggcagcca
     1321 tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt gtcgctcaag
     1381 gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat
     1441 tctgaaatga gctgttgaca attaatcatc ggctcgtata atgtgtggaa ttgtgagcgg
     1501 ataacaattt cacacaggaa acagccagtc cgtttaggtg ttttcacgag cacttcacca
     1561 acaaggacca tagattatga aaatcgaaga acaccatcac catcaccatt ccagcggtaa
     1621 gcttatgtcc cctatactag gttattggaa aattaagggc cttgtgcaac ccactcgact
     1681 tcttttggaa tatcttgaag aaaaatatga agagcatttg tatgagcgcg atgaaggtga
     1741 taaatggcga aacaaaaagt ttgaattggg tttggagttt cccaatcttc cttattatat
     1801 tgatggtgat gttaaattaa cacagtctat ggccatcata cgttatatag ctgacaagca
     1861 caacatgttg ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt
     1921 tttggatatt agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa
     1981 agttgatttt cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca
     2041 taaaacatat ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt atgacgctct
     2101 tgatgttgtt ttatacatgg acccaatgtg cctggatgcg ttcccaaaat tagtttgttt
     2161 taaaaaacgt attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat
     2221 agcatggcct ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaatc
     2281 ggatctggaa gttctgttcc aggggcccct gtccagcggt ctggttccgc gtggttccgg
     2341 taccgcggcc cagccggccg cagtccgtcg tgaagcagaa cgtcaagcac aagcccaact
     2401 ggaaaaagca aaaacgaaac cggtcgcatt cgcagtccgc accaacgtca gctattctgc
     2461 ggcccatgaa gatgacgtgc cggttccggg catggcaatt agttttgaag cgaaagattt
     2521 cctgcacgtt aaagcggccg cataataaac cgatacaatt aaaggctcct tttggagcct
     2581 tttttttgga gattttcaac gtgaaaaaat tattattcgc aattccttta gttgttcctt
     2641 tctattctca ctccgctgaa actgttgaaa gttgtttagc aaaaccccat acagaaaatt
     2701 catttactaa cgtctggaaa gacgacaaaa ctttagatcg ttacgctaac tatgagggct
     2761 gtctgtggaa tgctacaggc gttgtagttt gtactggtga cgaaactcag tgtctagcta
     2821 gagtggcggt ggctctggtt ccggtgattt tgattatgaa aagatggcaa acgctaataa
     2881 gggggctatg accgaaaatg ccgatgaaaa cgcgctacag tctgacgcta aaggcaaact
     2941 tgattctgtc gctactgatt acggtgctgc tatcgatggt ttcattggtg acgtttccgg
     3001 ccttgctaat ggtaatggtg ctactggtga ttttgctggc tctaattccc aaatggctca
     3061 agtcggtgac ggtgataatt cacctttaat gaataatttc cgtcaatatt taccttccct
     3121 ccctcaatcg gttgaatgtc gcccttttgt ctttggcgct ggtaaaccat atgaattttc
     3181 tattgattgt gacaaaataa acttattccg tggtgtcttt gcgtttcttt tatatgttgc
     3241 cacctttatg tatgtatttt ctacgtttgc taacatactg cgtaataagg agtcttaatc
     3301 atgccagttc ttttggctag cgccgcccta taccttgtct gcctccccgc gttgcgtcgc
     3361 ggtgcatgga gccgggccac ctcgacctga atggaagccg gcggcacctc gctaacggat
     3421 tcaccactcc aagaattgga gccaatcaat tcttgcggag aactgtgaat gcgcaaacca
     3481 acccttggca gaacatatcc atcgcgtccg ccatctccag cagccgcacg cggcgcatct
     3541 cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc
     3601 ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa
     3661 cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg
     3721 gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc
     3781 ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga
     3841 agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt
     3901 gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga
     3961 gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg
     4021 aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc
     4081 agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct
     4141 gtgaatcgct tcacgaccac gctgatgagc tttaccgcag gatccgcgaa attgtaagcg
     4201 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     4261 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     4321 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     4381 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     4441 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     4501 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     4561 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     4621 ttaatgcgcc gctacagggc gcgtcgcgga tcctgcctcg cgcgtttcgg tgatgacggt
     4681 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     4741 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     4801 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
     4861 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     4921 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4981 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5041 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5101 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5161 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag ggtttccccc
     5221 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5281 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5341 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5401 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5461 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5521 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     5581 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5641 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5701 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5761 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5821 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5881 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5941 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6001 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6061 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6121 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6181 tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6241 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6301 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6361 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6421 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6481 ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6541 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6601 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6661 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6721 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6781 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6841 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
//
