LOCUS       sequence-353155-        6443 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in bacterial cells. FERM domain, M1-E346. Can be 
            biotinylated..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6443)
  AUTHORS   Gileadi, et al
  TITLE     TREAT-AD plasmids 2021
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6443)
  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            "unpublished"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-c023
FEATURES             Location/Qualifiers
     source          1..6443
                     /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             1164..1208
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1261..1278
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1331..1349)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1345..1392
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1429..1884
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1516..1535)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1726..1747
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1976..2791)
                     /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(1976..2791)
                     /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     2699..2718
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2783..2802
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2913..3501
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3402..3421
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3655..3672
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3687..3826
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3752..3771)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3912..3934
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3928..4119)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4928..6010)
                     /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
                     EACKAAVHNLLAQRVSGLIINYQLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5972..5991
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6011..6088)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6294..6313
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6397..6416
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6397..6415
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6416..6440
                     /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 taccattgag gtgcagcaga
     1021 tgaaggcaca ggcccgggag gagaagcacc agaagcagat ggagcgtgct atgctggaaa
     1081 atgagaagaa gaagcgtgaa atggcagaga aggagaaaga gaagattgaa cgggagaagg
     1141 aggagagcag taaaggtgga tacggcctga atgatatctt tgaagcgcag aagattgaat
     1201 ggcatgaatg ataaggatcc gaattcgagc tccgtcgaca agcttgcggc cgcactcgag
     1261 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     1321 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1381 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     1441 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1501 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1561 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1621 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1681 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1741 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1801 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1861 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1921 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     1981 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     2041 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     2101 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     2161 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     2221 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     2281 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     2341 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     2401 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     2461 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     2521 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     2581 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     2641 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     2701 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     2761 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     2821 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2881 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2941 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3001 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     3061 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3121 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3181 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3241 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3301 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3361 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3421 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3481 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3541 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3601 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3661 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3721 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3781 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     3841 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3901 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3961 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     4021 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     4081 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     4141 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     4201 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     4261 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     4321 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     4381 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     4441 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     4501 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     4561 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     4621 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     4681 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     4741 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     4801 aagtgcggcg acgatagtca tgtcccgcgc ccaccggaag gagctgactg ggttgaaggc
     4861 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     4921 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4981 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     5041 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     5101 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     5161 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     5221 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     5281 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     5341 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     5401 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     5461 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     5521 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     5581 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc tgatagttaa
     5641 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     5701 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     5761 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     5821 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     5881 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     5941 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     6001 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     6061 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     6121 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     6181 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     6241 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     6301 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     6361 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     6421 tgtgagcgga taacaattcc cct
//
