LOCUS       sequence-315905-        6939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of LASP1_LIM.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6939)
  AUTHORS   Teyra J, Kelil A, Jain S, Helmy M, Jajodia R, Hooda Y, Gu J, D'Cruz 
            AA, Nicholson SE, Min J, Sudol M, Kim PM, Bader GD, Sidhu SS
  TITLE     Large-scale survey and database of high affinity ligands for peptide
            recognition modules.
  JOURNAL   Mol Syst Biol. 2020 Dec;16(12):e9310. doi: 10.15252/msb.20199310.
  PUBMED    33438817
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"; journalName: "Mol Syst 
            Biol."; date: "2020-12"; volume: "16(12):e9310. doi"; pages: " 
            10.15252/msb.20199310"
COMMENT     Sequence Label: pHH0103_LASP1_LIM
FEATURES             Location/Qualifiers
     source          1..6939
                     /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)."
     primer_bind     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             138..155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             171..824
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     782..804
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             831..854
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             867..884
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      1093..1102
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     terminator      1134..1181
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2774..3229)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2911..2932)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3123..3142
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3271..3293)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3379..3519
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3434..3453
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3534..3551)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3705..4292)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4463..5254,5255..5323))
                     /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     5086..5105
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5324..5428)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5496..5514
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5537..5614
                     /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             5615..6697
                     /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(5634..5653)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga
      121 ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt atgtccccta
      181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
      841 tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc gcggcccagc
      901 cggccatgaa ccctaattgt gcacgttgcg gcaaaatcgt atatccgact gaaaaagtta
      961 actgcctgga taaattctgg cacaaggcgt gtttccactg cgaaacctgc aaaatgaccc
     1021 tgaacatgaa aaactataaa ggttacgaga agaaaccgta ctgtaacgcc cattacccaa
     1081 aacagtcttt taccatggtg gctgacacgc cggaaaacgc ggccgcataa taaaccgata
     1141 caattaaagg ctccttttgg agcctttttt ttggagattt tcaacgtgaa aaaattatta
     1201 ttcgcaattc ctttagttgt tcctttctat tctcactccg ctgaaactgt tgaaagttgt
     1261 ttagcaaaac cccatacaga aaattcattt actaacgtct ggaaagacga caaaacttta
     1321 gatcgttacg ctaactatga gggctgtctg tggaatgcta caggcgttgt agtttgtact
     1381 ggtgacgaaa ctcagtgtct agctagagtg gcggtggctc tggttccggt gattttgatt
     1441 atgaaaagat ggcaaacgct aataaggggg ctatgaccga aaatgccgat gaaaacgcgc
     1501 tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt gctgctatcg
     1561 atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact ggtgattttg
     1621 ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct ttaatgaata
     1681 atttccgtca atatttacct tccctccctc aatcggttga atgtcgccct tttgtctttg
     1741 gcgctggtaa accatatgaa ttttctattg attgtgacaa aataaactta ttccgtggtg
     1801 tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttctacg tttgctaaca
     1861 tactgcgtaa taaggagtct taatcatgcc agttcttttg gctagcgccg ccctatacct
     1921 tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga cctgaatgga
     1981 agccggcggc acctcgctaa cggattcacc actccaagaa ttggagccaa tcaattcttg
     2041 cggagaactg tgaatgcgca aaccaaccct tggcagaaca tatccatcgc gtccgccatc
     2101 tccagcagcc gcacgcggcg catctcgggc agcgttgggt cctggccacg ggtgcgcatg
     2161 atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag
     2221 aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc
     2281 tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag
     2341 tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt
     2401 ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg
     2461 tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt
     2521 tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc
     2581 atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt
     2641 aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg
     2701 gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac
     2761 cgcaggatcc gcgaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     2821 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     2881 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     2941 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     3001 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     3061 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     3121 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     3181 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcggatcctg
     3241 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     3301 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     3361 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     3421 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
     3481 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     3541 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3601 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3661 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3721 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3781 ctataaagat accagggttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3841 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3901 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3961 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4021 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4081 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4141 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4201 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4261 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4321 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4381 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4441 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4501 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4561 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4621 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4681 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4741 cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct
     4801 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4861 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4921 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4981 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5041 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac
     5101 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5161 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5221 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5281 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5341 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5401 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     5461 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     5521 gtcttcaaga attcccgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata
     5581 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     5641 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     5701 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     5761 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     5821 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     5881 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     5941 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     6001 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     6061 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     6121 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     6181 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     6241 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     6301 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     6361 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     6421 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     6481 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     6541 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     6601 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     6661 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     6721 agctcactca ttaggcacaa ttctcatgtt tgacagctta tcatcgactg cacggtgcac
     6781 caatgcttct ggcgtcaggc agccatcgga agctgtggta tggctgtgca ggtcgtaaat
     6841 cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg
     6901 acatcataac ggttctggca aatattctga aatgagctg
//
