LOCUS       pHBT-HRas174            5860 bp    DNA     circular SYN 09-OCT-2025
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5860)
  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..5860
                     /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=HRas1-174
                     /note="color: #a6acb3"
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPP"
     CDS             722..739
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     terminator      808..855
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
                     /note="color: #ffffff"
     rep_origin      892..1347
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     CDS             complement(1440..2255)
                     /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      2377..2965
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     misc_feature    3151..3293
                     /label=bom
                     /note="basis of mobility region from pBR322"
                     /note="color: #a6acb3"
     CDS             complement(3395..3586)
                     /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(4395..5477)
                     /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(5478..5555)
                     /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 atgacggaat ataagcttgt ggtggtgggc gccggcggtg tgggcaagag
      241 tgcgctgacc atccagctga tccagaacca ctttgtggac gaatacgacc ccactataga
      301 ggattcctac cggaagcagg tggtcattga tggggagacg tgcctgttgg acatcctgga
      361 taccgccggc caggaggagt acagcgccat gcgggaccag tacatgcgca ccggggaggg
      421 cttcctgtgt gtgtttgcca tcaacaacac caagtctttt gaggacatcc accagtacag
      481 ggagcagatc aaacgggtga aggactcgga tgacgtgccc atggtgctgg tggggaacaa
      541 gtgtgacctg gctgcacgca ctgtggaatc tcggcaggct caggacctcg cccgaagcta
      601 cggcatcccc tacatcgaga cctcggccaa gacccggcag ggagtggagg atgccttcta
      661 cacgttggtg cgtgagatcc ggcagcacaa gctgcggaag ctgaaccctc cttgactcga
      721 gcaccaccac caccaccact gagatccggc agcacaagct gcggaagctg aaccctcctt
      781 gactcgagca ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      841 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
      901 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      961 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1021 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1081 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1141 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1201 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1261 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1321 ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa
     1381 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     1441 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     1501 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     1561 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     1621 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     1681 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     1741 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     1801 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     1861 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     1921 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     1981 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     2041 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     2101 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     2161 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     2221 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     2281 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2341 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2401 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2461 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2521 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2581 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2641 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2701 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     2761 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     2821 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     2881 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     2941 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3001 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3061 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3121 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3181 gtgcggtatt tcacaccgca tatatggtgc actctcagta caatctgctc tgatgccgca
     3241 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3301 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3361 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3421 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     3481 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     3541 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     3601 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     3661 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     3721 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     3781 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     3841 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     3901 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     3961 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4021 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     4081 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     4141 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     4201 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     4261 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     4321 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     4381 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4441 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     4501 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     4561 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     4621 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     4681 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     4741 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     4801 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     4861 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     4921 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     4981 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     5041 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     5101 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     5161 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     5221 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     5281 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     5341 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     5401 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     5461 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     5521 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     5581 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     5641 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     5701 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     5761 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     5821 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
//
