LOCUS       sequence-200155-        5355 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression and purification of LIMKINASE BGL1 PDZ domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5355)
  AUTHORS   Tonikian R, Zhang Y, Sazinsky SL, Currell B, Yeh JH, Reva B, Held 
            HA, Appleton BA, Evangelista M, Wu Y, Xin X, Chan AC, Seshagiri S, 
            Lasky LA, Sander C, Boone C, Bader GD, Sidhu SS
  TITLE     A specificity map for the PDZ domain family.
  JOURNAL   PLoS Biol. 2008 Sep 30;6(9):e239. doi: 10.1371/journal.pbio.0060239.
  PUBMED    18828675
REFERENCE   2  (bases 1 to 5355)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS 
            Biol."; date: "2008-09-30"; volume: "6(9):e239. doi"; pages: " 
            10.1371/journal.pbio.0060239"
COMMENT     Sequence Label: LIMK1_PDZ_1
FEATURES             Location/Qualifiers
     source          1..5355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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)."
     CDS             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..759
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1223..1245)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1390..1408)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1476..1580
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1581..1649,1650..2441)
                     /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     complement(1799..1818)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2612..3200
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3101..3120
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3354..3371
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3444..3521
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3522..4604
                     /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(3541..3560)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4653..4658,4659..4676,4677..4683)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4691..4707
                     /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     4715..4731
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4715..4731
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4727..5254
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4747..4764)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4747..4763)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4756..4778)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctgga agttctgttc caggggcccc tgggatctac ccccgtcatc
      781 gagcagatcc tgcctgactc ccctggctcc cacctgcccc acaccgtcac cctggtgtcc
      841 atcccagcct catctcatgg caagcgtgga ctttcagtct ccattgaccc cccgcacggc
      901 ccaccgggct gtggcaccga gcactcacac accgtccgcg tccagggagt ggatccgggc
      961 tgcatgagcc cagatgtgaa gaattccatc cacgtcggag accggatctt ggaaatcaat
     1021 ggcacgccca tccgaaatgt gcccctggac gagattgacc tgctgattca ggaaaccagc
     1081 cgcctgctcc agctgaccct cgagcatgac cctcacgata cactgggcca cgggctgggg
     1141 cctgagacct gagtcgactc gagcggccgc atcgtgactg actgacgatc tgcctcgcgc
     1201 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     1261 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     1321 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat aattcttgaa
     1381 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     1441 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1501 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1561 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1621 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1681 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1741 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1801 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     1861 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     1921 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     1981 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2041 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2101 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     2161 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2221 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2281 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2341 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2401 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2461 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2521 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2581 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2641 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2701 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2761 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2821 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2881 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     2941 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3001 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3061 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3121 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3181 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3241 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3301 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3361 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     3421 gtatttcaca ccgcataaat tccgacacca tcgaatggcg caaaaccttt cgcggtatgg
     3481 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     3541 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     3601 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     3661 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     3721 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     3781 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     3841 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     3901 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     3961 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     4021 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     4081 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     4141 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     4201 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     4261 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     4321 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     4381 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     4441 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     4501 aaaccacgct agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     4561 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     4621 gtaagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     4681 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     4741 ggattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     4801 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     4861 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg cctggtttcc
     4921 ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat cttcctgagg ccgatactgt
     4981 cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg cccatctaca ccaacgtgac
     5041 ctatcccatt acggtcaatc cgccgtttgt tcccacggag aatccgacgg gttgttactc
     5101 gctcacattt aatgttgatg aaagctggct acaggaaggc cagacgcgaa ttatttttga
     5161 tggcgttgga attagcttat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc
     5221 atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa
     5281 ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata
     5341 ttctgaaatg agctg
//
