LOCUS       sequence-353214-        6318 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in bacterial cells. FERM domain, M1-A321..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6318)
  AUTHORS   Gileadi, et al
  TITLE     TREAT-AD plasmids 2021
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6318)
  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            "unpublished"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: RDXB-c004
FEATURES             Location/Qualifiers
     source          1..6318
                     /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             1136..1153
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1206..1224)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1220..1267
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1304..1759
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1391..1410)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1601..1622
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1851..2666)
                     /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(1851..2666)
                     /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     2574..2593
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2658..2677
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2788..3376
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3277..3296
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3530..3547
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3562..3701
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3627..3646)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3787..3809
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3803..3994)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4803..5885)
                     /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     5847..5866
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5886..5963)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6169..6188
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6272..6291
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6272..6290
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6291..6315
                     /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 ccgaaaccaa
      121 tcaacgtaag agtaactaca atggatgctg agctggaatt tgccattcag cccaatacaa
      181 ctggcaaaca actttttgac caggtggtga aaacagttgg tttgcgtgag gtctggtttt
      241 ttgggctgca gtatgtagac agcaaaggtt attctacatg gcttaaacta aataaaaagg
      301 taacacagca ggatgttaaa aaagagaatc ctttacagtt caagtttaga gctaaattct
      361 ttcctgaaga tgtttctgag gaattaattc aagaaataac ccagagactc ttcttcttgc
      421 aagttaaaga agccatctta aatgatgaga tatattgccc gccagaaact gcagttcttt
      481 tggcttccta tgctgtccaa gccaagtatg gagattacaa taaagagatt cataagccag
      541 gctacctggc taatgataga ctcctacccc agcgtgtatt ggaacaacac aaactaacaa
      601 aagaacagtg ggaagaaaga atacagaact ggcatgaaga acatagagga atgttaaggg
      661 aggattctat gatggaatac ctgaagattg cacaagatct agaaatgtat ggagtcaact
      721 attttgaaat aaaaaataaa aaaggaactg aattgtggct aggtgttgat gctttgggtc
      781 tgaatattta tgagcatgac gacaagttaa cacctaaaat tggttttccc tggagtgaaa
      841 tcagaaatat ttcatttaat gacaaaaaat ttgttataaa gccaatcgac aaaaaggcac
      901 ctgattttgt gttttatgca cctcgtctga gaatcaataa gaggattttg gccttatgta
      961 tgggaaacca tgaactatac atgcgaagaa ggaagcctga tactattgaa gtacaacaga
     1021 tgaaggctca ggctagggag gagaaacatc agaagcagtt ggaaagggca tgacagtaaa
     1081 ggtggatacg gatccgaatt cgagctccgt cgacaagctt gcggccgcac tcgagcacca
     1141 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     1201 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     1261 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     1321 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1381 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1441 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1501 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1561 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1621 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     1681 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     1741 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1801 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     1861 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     1921 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     1981 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     2041 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     2101 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     2161 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     2221 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     2281 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     2341 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     2401 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     2461 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     2521 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     2581 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     2641 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     2701 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2761 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2821 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2881 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     2941 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3001 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3061 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3121 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3181 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3241 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3301 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3361 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3421 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3481 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3541 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3601 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3661 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     3721 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3781 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3841 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     3901 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     3961 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     4021 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     4081 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4141 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     4201 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4261 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4321 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4381 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4441 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     4501 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4561 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4621 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     4681 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     4741 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     4801 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4861 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     4921 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     4981 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     5041 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     5101 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     5161 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     5221 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5281 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     5341 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5401 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5461 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5521 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5581 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5641 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     5701 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     5761 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     5821 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     5881 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     5941 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     6001 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     6061 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     6121 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     6181 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     6241 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     6301 gcggataaca attcccct
//
