LOCUS       sequence-213656-        7512 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of LRP5_Ldl-recept-b_2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7512)
  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 7512)
  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_LRP5_Ldl-recept-b_2
FEATURES             Location/Qualifiers
     source          1..7512
                     /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"
     terminator      1707..1754
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3347..3802)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3484..3505)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3696..3715
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3844..3866)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3952..4092
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4007..4026
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4107..4124)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4278..4865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5036..5827,5828..5896))
                     /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     5659..5678
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5897..6001)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6069..6087
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6110..6187
                     /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             6188..7270
                     /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(6207..6226)
                     /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 cggccgcaga agaagtactg ctgctggctc gtcgtaccga cctgcgtcgc atctccctgg
      961 acaccccgga ttttactgac attgttctgc aagtggatga tatccgtcac gcgatcgcaa
     1021 ttgactatga tccgctggaa ggttatgttt actggaccga cgacgaagta cgcgcgattc
     1081 gtcgtgcgta cctggacggt tccggcgcgc agaccctggt gaatactgag atcaacgacc
     1141 cggacggcat tgcagtggat tgggttgcac gcaacctgta ctggacggac acgggcactg
     1201 accgtatcga ggtgacccgc ctgaacggta ccagccgcaa aatcctggtt agcgaagatc
     1261 tggacgagcc gcgcgccatc gccctgcatc cggttatggg cctgatgtac tggactgatt
     1321 ggggtgaaaa cccgaaaatc gaatgcgcga atctggatgg ccaggaacgt cgtgtactgg
     1381 taaacgcttc tctgggctgg cctaacggtc tggccctgga tctgcaggaa ggtaagctgt
     1441 actggggcga cgctaaaacc gataaaatcg aagttatcaa cgtcgacggc accaagcgtc
     1501 gtactctgct ggaggataaa ctgccacaca tcttcggttt caccctgctg ggtgatttca
     1561 tttattggac cgattggcag cgtcgttcta tcgaacgtgt gcacaaagtt aaagcgtctc
     1621 gcgacgttat cattgatcag ctgccggacc tgatgggtct gaaagctgtt aacgtcgcta
     1681 aagtggtcgg tgcggccgca taataaaccg atacaattaa aggctccttt tggagccttt
     1741 tttttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc
     1801 tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aaccccatac agaaaattca
     1861 tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta tgagggctgt
     1921 ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg aaactcagtg tctagctaga
     1981 gtggcggtgg ctctggttcc ggtgattttg attatgaaaa gatggcaaac gctaataagg
     2041 gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc tgacgctaaa ggcaaacttg
     2101 attctgtcgc tactgattac ggtgctgcta tcgatggttt cattggtgac gtttccggcc
     2161 ttgctaatgg taatggtgct actggtgatt ttgctggctc taattcccaa atggctcaag
     2221 tcggtgacgg tgataattca cctttaatga ataatttccg tcaatattta ccttccctcc
     2281 ctcaatcggt tgaatgtcgc ccttttgtct ttggcgctgg taaaccatat gaattttcta
     2341 ttgattgtga caaaataaac ttattccgtg gtgtctttgc gtttctttta tatgttgcca
     2401 cctttatgta tgtattttct acgtttgcta acatactgcg taataaggag tcttaatcat
     2461 gccagttctt ttggctagcg ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg
     2521 tgcatggagc cgggccacct cgacctgaat ggaagccggc ggcacctcgc taacggattc
     2581 accactccaa gaattggagc caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac
     2641 ccttggcaga acatatccat cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg
     2701 ggcagcgttg ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg
     2761 ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg
     2821 tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt
     2881 ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg
     2941 atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag
     3001 cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt
     3061 ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc
     3121 atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag
     3181 gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag
     3241 acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt
     3301 gaatcgcttc acgaccacgc tgatgagctt taccgcagga tccgcgaaat tgtaagcgtt
     3361 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     3421 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     3481 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     3541 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     3601 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     3661 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     3721 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     3781 aatgcgccgc tacagggcgc gtcgcggatc ctgcctcgcg cgtttcggtg atgacggtga
     3841 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     3901 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     3961 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     4021 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     4081 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4141 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4201 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4261 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4321 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggg tttccccctg
     4381 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4441 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4501 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4561 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4621 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4681 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     4741 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4801 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4861 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4921 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4981 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5041 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5101 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5161 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5221 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5281 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5341 ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5401 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5461 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5521 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5581 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5641 gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     5701 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5761 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5821 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5881 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5941 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6001 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     6061 cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattcccg acaccatcga
     6121 atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
     6181 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
     6241 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
     6301 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
     6361 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
     6421 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
     6481 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
     6541 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
     6601 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
     6661 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
     6721 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
     6781 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
     6841 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
     6901 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
     6961 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
     7021 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     7081 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     7141 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
     7201 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     7261 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca caattctcat
     7321 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
     7381 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
     7441 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
     7501 tgaaatgagc tg
//
