LOCUS       pHBT-NRas174            5858 bp    DNA     circular SYN 09-OCT-2025
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5858)
  AUTHORS   Koide Lab
  TITLE     Direct Submission
  JOURNAL   Exported Oct 9, 2025 from SnapGene 8.1.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        4..22
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     protein_bind    23..47
                     /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)."
                     /note="color: #31849b"
     RBS             62..84
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
                     /note="color: #a6acb3"
     CDS             98..115
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     CDS             122..166
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /note="color: #cc99b2"
                     /translation="GLNDIFEAQKIEWHE"
     CDS             167..187
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /note="color: #cc99b2
                     Cleavage site after base 184"
                     /translation="ENLYFQG"
     CDS             191..715
                     /codon_start=1
                     /label=NRas1-174
                     /note="color: #a6acb3"
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVK
                     DSDDVPMVLVGNKCDLPTRTVDTKQAHELAKSYGIPFIETSAKTRQGVEDAFYTLVREI
                     RQYRMKKLNSS"
     misc_feature    341..343
                     /label=C51
                     /note="color: #a6acb3"
     misc_feature    428..430
                     /label=C80
                     /note="color: #a6acb3"
     misc_feature    542..544
                     /label=C118
                     /note="color: #a6acb3"
     CDS             722..739
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     terminator      806..853
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
                     /note="color: #ffffff"
     rep_origin      890..1345
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     CDS             complement(1438..2253)
                     /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"
                     /note="color: #ccffcc"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      2375..2963
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     misc_feature    3149..3291
                     /label=bom
                     /note="basis of mobility region from pBR322"
                     /note="color: #a6acb3"
     CDS             complement(3393..3584)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /note="color: #993366"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4393..5475)
                     /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)."
                     /note="color: #993366"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(5476..5553)
                     /gene="lacI"
                     /label=lacI promoter
                     /note="color: #ffffff; direction: LEFT"
ORIGIN
        1 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
       61 ttttgtttaa ctttaagaag gagatataca tatgaaacat catcatcatc atcacagcag
      121 cggcctgaat gatatttttg aagcacaaaa aattgaatgg catgaagaaa acctgtactt
      181 ccagggatcc atgactgagt acaaactggt ggtggttgga gcaggtggtg ttgggaaaag
      241 cgcactgaca atccagctaa tccagaacca ctttgtagat gaatatgatc ccaccataga
      301 ggattcttac agaaaacaag tggttataga tggtgaaacc tgtttgttgg acatactgga
      361 tacagctgga caagaagagt acagtgccat gagagaccaa tacatgagga caggcgaagg
      421 cttcctctgt gtatttgcca tcaataatag caagtcattt gcggatatta acctctacag
      481 ggagcagatt aagcgagtaa aagactcgga tgatgtacct atggtgctag tgggaaacaa
      541 gtgtgatttg ccaacaagga cagttgatac aaaacaagcc cacgaactgg ccaagagtta
      601 cgggattcca ttcattgaaa cctcagccaa gaccagacag ggtgttgaag atgcttttta
      661 cacactggta agagaaatac gccagtaccg aatgaaaaaa ctcaacagca gttgactcga
      721 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
      781 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
      841 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
      901 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
      961 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     1021 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     1081 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     1141 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     1201 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     1261 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     1321 aacaaaatat taacgtttac aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc
     1381 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     1441 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     1501 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     1561 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     1621 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     1681 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     1741 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     1801 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     1861 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     1921 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     1981 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     2041 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     2101 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     2161 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     2221 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     2281 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     2341 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     2401 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     2461 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     2521 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     2581 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     2641 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     2701 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     2761 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     2821 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2881 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2941 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3001 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3061 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3121 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3181 gcggtatttc acaccgcata tatggtgcac tctcagtaca atctgctctg atgccgcata
     3241 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3301 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3361 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3421 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     3481 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     3541 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     3601 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     3661 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     3721 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     3781 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     3841 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     3901 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     3961 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     4021 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     4081 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     4141 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     4201 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     4261 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     4321 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     4381 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4441 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     4501 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     4561 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     4621 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     4681 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     4741 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     4801 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     4861 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     4921 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     4981 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     5041 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     5101 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     5161 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     5221 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     5281 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     5341 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     5401 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     5461 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     5521 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     5581 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     5641 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     5701 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     5761 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     5821 tgcgtccggc gtagaggatc gagatctcga tcccgcga
//
