LOCUS       sequence-353162-        5868 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in bacterial cells. GTPase domain, G15-N184. Can 
            be used for crystallography..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5868)
  AUTHORS   Gileadi, et al
  TITLE     TREAT-AD plasmids 2021
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5868)
  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            "unpublished"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: RAB5AA-c006
FEATURES             Location/Qualifiers
     source          1..5868
                     /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             686..703
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(756..774)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      770..817
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      854..1309
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(941..960)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1151..1172
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1401..2216)
                     /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(1401..2216)
                     /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     2124..2143
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2208..2227
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2338..2926
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2827..2846
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3080..3097
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3112..3251
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3177..3196)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3337..3359
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3353..3544)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4353..5435)
                     /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     5397..5416
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5436..5513)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5719..5738
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5822..5841
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5822..5840
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5841..5865
                     /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 ggaaataaaa
      121 tatgccagtt caaactagta cttctgggag agtccgctgt tggcaaatca agcctagtgc
      181 ttcgttttgt gaaaggccaa tttcatgaat ttcaagagag taccattggg gctgcttttc
      241 taacccaaac tgtatgtctt gatgacacta cagtaaagtt tgaaatatgg gatacagctg
      301 gtcaagaacg ataccatagc ctagcaccaa tgtactacag aggagcacaa gcagccatag
      361 ttgtatatga tatcacaaat gaggagtcct ttgcaagagc aaaaaattgg gttaaagaac
      421 ttcagaggca agcaagtcct aacattgtaa tagctttatc gggaaacaag gccgacctag
      481 caaataaaag agcagtagat ttccaggaag cacagtccta tgcagatgac aatagtttat
      541 tattcatgga gacatccgct aaaacatcaa tgaatgtaaa tgaaatattc atggcaatag
      601 ctaaaaaatt gccaaagaat tgacagtaaa ggtggatacg gatccgaatt cgagctccgt
      661 cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
      721 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
      781 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
      841 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
      901 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
      961 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1021 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1081 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1141 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     1201 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     1261 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     1321 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1381 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     1441 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     1501 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     1561 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     1621 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     1681 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     1741 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     1801 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     1861 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     1921 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     1981 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     2041 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     2101 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     2161 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     2221 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     2281 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     2341 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     2401 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     2461 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     2521 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     2581 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     2641 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     2701 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2761 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2821 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2881 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2941 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3001 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3061 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     3121 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     3181 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     3241 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3301 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3361 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     3421 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     3481 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     3541 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     3601 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     3661 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     3721 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     3781 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     3841 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     3901 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     3961 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     4021 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     4081 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     4141 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     4201 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     4261 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     4321 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4381 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4441 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     4501 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     4561 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     4621 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     4681 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     4741 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     4801 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     4861 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     4921 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     4981 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5041 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5101 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     5161 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     5221 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     5281 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     5341 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     5401 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     5461 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     5521 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     5581 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     5641 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     5701 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     5761 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     5821 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccct
//
