LOCUS       sequence-353157-        6393 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression in bacterial cells. FERM domain, M1-E346. 
            Contains L281R point mutation that reduces binding to CD44..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6393)
  AUTHORS   Du Y, Bradshaw WJ, Leisner TM, Annor-Gyamfi JK, Qian K, Bashore FM, 
            Sikdar A, Nwogbo FO, Ivanov AA, Frye SV, Gileadi O, Brennan PE, 
            Levey AI, Axtman AD, Pearce KH, Fu H, Katis VL
  TITLE     Discovery of FERM domain protein-protein interaction inhibitors for 
            MSN and CD44 as a potential therapeutic approach for Alzheimer's 
            disease.
  JOURNAL   J Biol Chem. 2023 Oct 20:105382. doi: 10.1016/j.jbc.2023.105382.
  PUBMED    37866628
REFERENCE   2  (bases 1 to 6393)
  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: "J Biol 
            Chem. 2023 Oct 20:105382. doi: 10.1016/j.jbc.2023.105382."
COMMENT     Sequence Label: MSNA-c021
FEATURES             Location/Qualifiers
     source          1..6393
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             12..34
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             45..62
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             87..107
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1211..1228
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1281..1299)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1295..1342
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1379..1834
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1466..1485)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1676..1697
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1926..2741)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1926..2741)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2649..2668
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2733..2752
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2863..3451
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3352..3371
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3605..3622
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3637..3776
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3702..3721)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3862..3884
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3878..4069)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4878..5960)
                     /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     5922..5941
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5961..6038)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6244..6263
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6347..6366
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6347..6365
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6366..6390
                     /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)."
ORIGIN
        1 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgcaccat catcatcatc
       61 attcttctgg tgtagatctg ggtaccgaga acctgtactt ccaatccatg cccaaaacga
      121 tcagtgtgcg tgtgaccacc atggatgcag agctggagtt tgccatccag cccaacacca
      181 ccgggaagca gctatttgac caggtggtga aaactattgg cttgagggaa gtttggttct
      241 ttggtctgca gtaccaggac actaaaggtt tctccacctg gctgaaactc aataagaagg
      301 tgactgccca ggatgtgcgg aaggaaagcc ccctgctctt taagttccgt gccaagttct
      361 accctgagga tgtgtccgag gaattgattc aggacatcac tcagcgcctg ttctttctgc
      421 aagtgaaaga gggcattctc aatgatgata tttactgccc gcctgagacc gctgtgctgc
      481 tggcctcgta tgctgtccag tctaagtatg gcgacttcaa taaggaagtg cataagtctg
      541 gctacctggc cggagacaag ttgctcccgc agagagtcct ggaacagcac aaactcaaca
      601 aggaccagtg ggaggagcgg atccaggtgt ggcatgagga acaccgtggc atgctcaggg
      661 aggatgctgt cctggaatat ctgaagattg ctcaagatct ggagatgtat ggtgtgaact
      721 acttcagcat caagaacaag aaaggctcag agctgtggct gggggtggat gccctgggtc
      781 tcaacatcta tgagcagaat gacagactaa ctcccaagat aggcttcccc tggagtgaaa
      841 tcaggaacat ctctttcaat gataagaaat ttgtcatcaa gcccattgac aaaaaagccc
      901 cggacttcgt cttctatgct ccccggctaa gaatcaataa aagaattagg gccttgtgca
      961 tggggaacca tgaactatac atgcgccgtc gcaagcctga taccattgag gtgcagcaga
     1021 tgaaggcaca ggcccgggag gagaagcacc agaagcagat ggagcgtgct atgctggaaa
     1081 atgagaagaa gaagcgtgaa atggcagaga aggagaaaga gaagattgaa cgggagaagg
     1141 aggagtgaca gtaaaggtgg atacggatcc gaattcgagc tccgtcgaca agcttgcggc
     1201 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     1261 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1321 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     1381 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1441 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1501 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1561 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     1621 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1681 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     1741 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     1801 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     1861 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     1921 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     1981 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2041 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2101 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2161 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     2221 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2281 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2341 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2401 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     2461 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2521 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2581 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     2641 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     2701 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     2761 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     2821 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2881 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2941 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3001 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3061 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3121 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3181 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3241 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3301 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3361 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3421 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3481 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3541 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3601 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3661 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     3721 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     3781 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3841 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3901 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     3961 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     4021 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     4081 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     4141 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     4201 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     4261 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     4321 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     4381 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     4441 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     4501 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     4561 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     4621 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     4681 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     4741 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     4801 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     4861 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4921 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     4981 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     5041 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     5101 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     5161 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     5221 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     5281 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     5341 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     5401 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     5461 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     5521 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     5581 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     5641 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     5701 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     5761 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     5821 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     5881 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     5941 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     6001 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     6061 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     6121 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     6181 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     6241 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     6301 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
     6361 ataggggaat tgtgagcgga taacaattcc cct
//
