LOCUS       sequence-411209-        5957 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RHOA protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5957)
  TITLE     TREAT-AD plasmids Jun 2023
REFERENCE   2  (bases 1 to 5957)
  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"
COMMENT     Sequence Label: RHOAA-c035
FEATURES             Location/Qualifiers
     source          1..5957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     907..926
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(946..1023)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1229..1248
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1332..1351
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1332..1350
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1351..1375
                     /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)."
     RBS             1390..1412
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1423..1440
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1465..1485
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2056..2100
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2153..2170
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2223..2241)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2237..2284
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2321..2776
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2408..2427)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2618..2639
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2868..3683)
                     /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(2868..3683)
                     /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     3591..3610
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3675..3694
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3805..4393
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4294..4313
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4547..4564
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4579..4718
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4644..4663)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4804..4826
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4820..5011)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5820..5957,1..945))
                     /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"
ORIGIN
        1 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
       61 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
      121 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
      181 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
      241 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
      301 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
      361 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
      421 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
      481 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
      541 agcaatggca tcctggtcat ccagctgata gttaatgatc agcccactga cgcgttgcgc
      601 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
      661 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
      721 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
      781 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
      841 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
      901 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
      961 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     1021 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     1081 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     1141 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     1201 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     1261 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     1321 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     1381 agaaataatt ttgtttaact ttaagaagga gatatacata tgcaccatca tcatcatcat
     1441 tcttctggtg tagatctggg taccgagaac ctgtacttcc aatccatggc tgcaattagg
     1501 aagaaactag taatagtggg cgacggcgcg tgtggtaaga cctgcttgct gattgttttc
     1561 tcgaaagatc agtttccgga ggtctatgtg cctacggttt tcgagaacta cgtggcggat
     1621 atcgaagtcg atggtaaaca ggttgaactg gcactgtggg ataccgcggg tcaggaggac
     1681 tacgaccgtc tgcgtccgct gtcctacccg gacaccgatg ttattctgat gtgctttagc
     1741 attgatagcc cagacagctt ggaaaacatc ccggagaaat ggacgccgga ggttaaacac
     1801 ttctgcccga atgtgccgat catcctcgta ggtaacaaga aggacctgcg caacgatgaa
     1861 catactagac gtgagctggc gaagatgaaa caagaaccgg ttaagccgga ggaaggccgt
     1921 gacatggcga atcgtattgg tgcctttggt tatatggaat gttctgctaa gaccaaagac
     1981 ggcgttcgcg aggtgttcga aatggctacc cgtgcggcat tacaagcccg tcgcggtagc
     2041 agtaaaggtg gatacggcct gaatgatatc tttgaagcgc agaagattga atggcatgaa
     2101 tgataaggat ccgaattcga gctccgtcga caagcttgcg gccgcactcg agcaccacca
     2161 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     2221 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     2281 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     2341 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2401 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2461 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2521 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2581 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2641 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2701 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     2761 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2821 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     2881 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     2941 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     3001 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     3061 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     3121 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     3181 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     3241 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     3301 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     3361 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     3421 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     3481 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     3541 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     3601 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     3661 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     3721 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3781 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3841 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3901 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3961 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4021 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4081 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4141 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4201 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4261 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4321 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4381 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4441 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4501 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4561 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4621 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     4681 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4741 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4801 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4861 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     4921 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     4981 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     5041 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     5101 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     5161 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     5221 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     5281 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     5341 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     5401 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     5461 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     5521 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     5581 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     5641 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     5701 cgacgatagt catgtcccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     5761 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     5821 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5881 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     5941 acgggcaaca gctgatt
//
