LOCUS       sequence-192406-        6939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            CACNB2-2/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6939)
  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 6939)
  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_CACNB2-2/2
FEATURES             Location/Qualifiers
     source          1..6939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..40
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        63..140
                     /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             141..1223
                     /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(160..179)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1466..1471,1472..1487,1488..1494)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1502..1518
                     /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     1507..1529
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1603..1620
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1636..2289
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2247..2269
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2296..2319
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2332..2349
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2599..2646
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4239..4694)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4376..4397)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4588..4607
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4736..4758)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4844..4984
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4899..4918
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4999..5016)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5170..5757)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5928..6719,6720..6788))
                     /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     6551..6570
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6789..6893)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaattc
       61 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
      121 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      181 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      241 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      301 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      361 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      421 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      481 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      541 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      601 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      661 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      721 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      781 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      841 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      901 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
      961 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
     1021 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1081 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1141 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1201 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1261 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1321 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1381 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1441 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1501 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1561 gcacttcacc aacaaggacc atagattatg aaaatcgaag aacaccatca ccatcaccat
     1621 tccagcggta agcttatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
     1681 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
     1741 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
     1801 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
     1861 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
     1921 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
     1981 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
     2041 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
     2101 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
     2161 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
     2221 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
     2281 cctccaaaat cggatctgga agttctgttc caggggcccc tgtccagcgg tctggttccg
     2341 cgtggttccg gtaccgcggc ccagccggcc atggcccagg gcgcgctgtt tgccgttcgt
     2401 accaacgttt cttattctgc cgcccatgaa gatgatgttc cagttccggg tatggccatt
     2461 tccttcgaag ccaaagattt cctgcatgtc aaagaaaaat ttaacaacga ttggtggatt
     2521 ggtcgtctgg ttaaagaagg ttgtgaaatt ggtttcattc cgtccccggt caaactggaa
     2581 aacgcggccg cataataaac cgatacaatt aaaggctcct tttggagcct tttttttgga
     2641 gattttcaac gtgaaaaaat tattattcgc aattccttta gttgttcctt tctattctca
     2701 ctccgctgaa actgttgaaa gttgtttagc aaaaccccat acagaaaatt catttactaa
     2761 cgtctggaaa gacgacaaaa ctttagatcg ttacgctaac tatgagggct gtctgtggaa
     2821 tgctacaggc gttgtagttt gtactggtga cgaaactcag tgtctagcta gagtggcggt
     2881 ggctctggtt ccggtgattt tgattatgaa aagatggcaa acgctaataa gggggctatg
     2941 accgaaaatg ccgatgaaaa cgcgctacag tctgacgcta aaggcaaact tgattctgtc
     3001 gctactgatt acggtgctgc tatcgatggt ttcattggtg acgtttccgg ccttgctaat
     3061 ggtaatggtg ctactggtga ttttgctggc tctaattccc aaatggctca agtcggtgac
     3121 ggtgataatt cacctttaat gaataatttc cgtcaatatt taccttccct ccctcaatcg
     3181 gttgaatgtc gcccttttgt ctttggcgct ggtaaaccat atgaattttc tattgattgt
     3241 gacaaaataa acttattccg tggtgtcttt gcgtttcttt tatatgttgc cacctttatg
     3301 tatgtatttt ctacgtttgc taacatactg cgtaataagg agtcttaatc atgccagttc
     3361 ttttggctag cgccgcccta taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga
     3421 gccgggccac ctcgacctga atggaagccg gcggcacctc gctaacggat tcaccactcc
     3481 aagaattgga gccaatcaat tcttgcggag aactgtgaat gcgcaaacca acccttggca
     3541 gaacatatcc atcgcgtccg ccatctccag cagccgcacg cggcgcatct cgggcagcgt
     3601 tgggtcctgg ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg
     3661 gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga
     3721 ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt
     3781 ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat
     3841 cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca
     3901 ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc
     3961 acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc
     4021 tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt
     4081 gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac
     4141 gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct
     4201 tcacgaccac gctgatgagc tttaccgcag gatccgcgaa attgtaagcg ttaatatttt
     4261 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     4321 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     4381 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     4441 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     4501 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     4561 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     4621 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     4681 gctacagggc gcgtcgcgga tcctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     4741 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     4801 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     4861 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     4921 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     4981 caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5041 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5101 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5161 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5221 tcagaggtgg cgaaacccga caggactata aagataccag ggtttccccc tggaagctcc
     5281 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5341 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5401 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5461 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5521 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5581 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     5641 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5701 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5761 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5821 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5881 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5941 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6001 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6061 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6121 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6181 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6241 gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6301 caacgatcaa ggcgagttac atgatccccc atattgtgca aaaaagcggt tagctccttc
     6361 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6421 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6481 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6541 tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6601 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6661 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6721 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6781 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6841 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6901 ccccgaaaag tgccacctga cgtctaagaa accattatt
//
