LOCUS       sequence-136222-        5911 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination; may not be full 
            ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5911)
  TITLE     SGC Oxford
REFERENCE   2  (bases 1 to 5911)
  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: RHOA
FEATURES             Location/Qualifiers
     source          1..5911
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             772..789
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(842..860)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      856..903
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      940..1395
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1027..1046)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1237..1258
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1488..2303)
                     /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(1488..2303)
                     /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     2211..2230
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2295..2314
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2425..3013
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2914..2933
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3167..3184
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3199..3341
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3267..3286)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3427..3449
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3443..3634)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4443..5525)
                     /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     5487..5506
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5526..5603)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5809..5828
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatggct gccatccgga agaaactggt
      181 gattgttggt gatggagcct gtggaaagac atgcttgctc atagtcttca gcaaggacca
      241 gttcccagag gtgtatgtgc ccacagtgtt tgagaactat gtggcagata tcgaggtgga
      301 tggaaagcag gtagaattgg ctttgtggga cacagctggg caggaagatt atgatcgcct
      361 gaggcccctc tcctacccag ataccgatgt tatactgatg tgtttttcca tcgacagccc
      421 tgatagttta gaaaacatcc cagaaaagtg gaccccagaa gtcaagcatt tctgtcccaa
      481 cgtgcccatc atcctggttg ggaataagaa ggatcttcgg aatgatgagc acacaaggcg
      541 ggagctagcc aagatgaagc aggagccggt gaaacctgaa gaaggcagag atatggcaaa
      601 caggattggc gcttttgggt acatggagtg ttcagcaaag accaaagatg gagtgagaga
      661 ggtttttgaa atggctacga gagctgctct gcaagctaga cgtgggtgac agtaaaggtg
      721 gatacggatc cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac
      781 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
      841 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      901 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
      961 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1021 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1081 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1141 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1201 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1261 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1321 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1381 taacgtttac aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1441 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     1501 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     1561 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     1621 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     1681 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     1741 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     1801 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     1861 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     1921 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     1981 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     2041 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     2101 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     2161 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     2221 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     2281 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     2341 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2401 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2461 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2521 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     2581 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2641 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2701 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2761 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2821 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2881 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2941 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3001 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3061 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3121 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3181 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3241 acaccgcata tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3301 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     3361 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3421 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3481 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     3541 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     3601 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     3661 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     3721 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     3781 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     3841 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     3901 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     3961 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4021 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4081 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     4141 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     4201 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     4261 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     4321 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     4381 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     4441 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4501 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     4561 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     4621 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     4681 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     4741 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     4801 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     4861 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     4921 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     4981 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5041 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5101 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5161 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5221 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5281 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     5341 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     5401 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     5461 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     5521 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     5581 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     5641 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     5701 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     5761 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     5821 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     5881 gtagaggatc gagatctcga tcccgcgaaa t
//
