LOCUS       sequence-353148-        7086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in bacterial cells. Entire CDS, M1-M577.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7086)
  AUTHORS   Gileadi, et al
  TITLE     TREAT-AD plasmids 2021
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 7086)
  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            "unpublished"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-c000
FEATURES             Location/Qualifiers
     source          1..7086
                     /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             1904..1921
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1974..1992)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1988..2035
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2072..2527
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2159..2178)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2369..2390
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2619..3434)
                     /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(2619..3434)
                     /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     3342..3361
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3426..3445
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3556..4144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4045..4064
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4298..4315
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4330..4469
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4395..4414)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4555..4577
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4571..4762)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5571..6653)
                     /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     6615..6634
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6654..6731)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6937..6956
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7040..7059
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7040..7058
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7059..7083
                     /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 aggagctgat ggagaggctg aagcagatcg aggaacagac taagaaggct cagcaagaac
     1201 tggaagaaca gacccgtagg gctctggaac ttgagcagga acggaagcgt gcccagagcg
     1261 aggctgaaaa gctggccaag gagcgtcaag aagctgaaga ggccaaggag gccttgctgc
     1321 aggcctcccg ggaccagaaa aagactcagg aacagctggc cttggaaatg gcagagctga
     1381 cagctcgaat ctcccagctg gagatggccc gacagaagaa ggagagtgag gctgtggagt
     1441 ggcagcagaa ggcccagatg gtacaggaag acttggagaa gacccgtgct gagctgaaga
     1501 ctgccatgag tacacctcat gtggcagagc ctgctgagaa tgagcaggat gagcaggatg
     1561 agaatggggc agaggctagt gctgacctac gggctgatgc tatggccaag gaccgcagtg
     1621 aggaggaacg taccactgag gcagagaaga atgagcgtgt gcagaagcac ctgaaggccc
     1681 tcacttcgga gctggccaat gccagagatg agtccaagaa gactgccaat gacatgatcc
     1741 atgctgagaa catgcgactg ggccgagaca aatacaagac cctgcgccag atccggcagg
     1801 gcaacaccaa gcagcgcatt gacgaatttg agtctatgtg acagtaaagg tggatacgga
     1861 tccgaattcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca
     1921 ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     1981 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     2041 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     2101 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2161 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2221 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2281 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2341 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2401 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2461 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     2521 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2581 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
     2641 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     2701 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     2761 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     2821 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     2881 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     2941 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     3001 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     3061 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     3121 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     3181 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     3241 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     3301 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     3361 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     3421 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     3481 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3541 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3601 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3661 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     3721 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3781 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3841 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3901 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3961 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     4021 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     4081 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     4141 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     4201 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4261 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     4321 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     4381 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     4441 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     4501 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4561 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     4621 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     4681 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     4741 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     4801 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     4861 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     4921 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     4981 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     5041 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     5101 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     5161 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     5221 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     5281 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     5341 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     5401 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     5461 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     5521 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     5581 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5641 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     5701 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     5761 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     5821 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     5881 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     5941 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     6001 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     6061 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     6121 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     6181 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     6241 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     6301 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     6361 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     6421 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     6481 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     6541 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     6601 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     6661 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     6721 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     6781 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     6841 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     6901 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     6961 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     7021 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     7081 tcccct
//
