LOCUS       sequence-353136-        6249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in bacterial cells. FERM domain, M1-D298. Used 
            for NMR studies..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6249)
  AUTHORS   Gileadi, et al
  TITLE     TREAT-AD plasmids 2021
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6249)
  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: 
            "unpublished"
COMMENT     Sequence Label: MSNA-c007
FEATURES             Location/Qualifiers
     source          1..6249
                     /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             1067..1084
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1137..1155)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1151..1198
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1235..1690
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1322..1341)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1532..1553
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1782..2597)
                     /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(1782..2597)
                     /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     2505..2524
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2589..2608
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2719..3307
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3208..3227
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3461..3478
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3493..3632
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3558..3577)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3718..3740
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3734..3925)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4734..5816)
                     /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     5778..5797
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5817..5894)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6100..6119
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6203..6222
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6203..6221
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6222..6246
                     /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 ccccggctgc ggattaacaa gcggatcttg gccttgtgca
      961 tggggaacca tgaactatac atgcgccgtc gcaagcctga ttgacagtaa aggtggatac
     1021 ggatccgaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca
     1081 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1141 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1201 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1261 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1321 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1381 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1441 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1501 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1561 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1621 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1681 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1741 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     1801 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     1861 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     1921 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     1981 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2041 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2101 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     2161 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2221 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2281 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2341 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2401 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2461 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2521 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     2581 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     2641 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2701 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2761 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2821 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     2881 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2941 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3001 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3061 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3121 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3181 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3241 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3301 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3361 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3421 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3481 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3541 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3601 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3661 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3721 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     3781 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     3841 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     3901 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     3961 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4021 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4081 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4141 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4201 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4261 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4321 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4381 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     4441 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     4501 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     4561 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     4621 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     4681 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     4741 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4801 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     4861 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     4921 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     4981 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     5041 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5101 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     5161 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     5221 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5281 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5341 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5401 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5461 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5521 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5581 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     5641 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     5701 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     5761 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     5821 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     5881 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     5941 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     6001 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     6061 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     6121 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     6181 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     6241 aattcccct
//
