LOCUS       sequence-423511-        5888 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of HaloTag fused to the C-terminal tail of 
            KRas4B.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5888)
  AUTHORS   Ajiki M, Yoshikawa M, Miyazaki T, Kawasaki A, Aoki K, Nakatsu F, 
            Tsukiji S
  TITLE     ORP9-PH domain-based fluorescent reporters for visualizing 
            phosphatidylinositol 4-phosphate dynamics in living cells.
  JOURNAL   RSC Chem Biol. 2024 Apr 24;5(6):544-555. doi: 10.1039/d3cb00232b. 
            eCollection 2024 Jun 5.
  PUBMED    38846081
REFERENCE   2  (bases 1 to 5888)
  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"; doi: 
            "10.1039/d3cb00232b"; journalName: "RSC Chem Biol"; date: 
            "2024-04-24- 24"; volume: "5"; issue: "6"; pages: "544-555"
COMMENT     Sequence Label: pCAGGS-HaloTag-KRas4B(CT)
FEATURES             Location/Qualifiers
     source          1..5888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(103..691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(183..202)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(862..1653,1654..1722))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1485..1504
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1723..1827)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1858..2237
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2239..2514
                     /label=chicken beta-actin promoter
     intron          2515..3531
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3455..3477
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3539..3558
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3586..4476
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     primer_bind     complement(4606..4626)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4606..4624)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4645..4661)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4763..4782)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4828..4883
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4882..4901)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5244..5260)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5268..5284
                     /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)."
     promoter        complement(join(5292..5298,5299..5316,5317..5322))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5337..5358
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5417..5612
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5463..5598
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5618..5752
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5655..5674)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5709..5728
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5820..5837)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
       61 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
      121 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
      181 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
      241 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
      301 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
      361 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
      421 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
      481 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
      541 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
      601 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
      661 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
      721 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
      781 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
      841 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
      901 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
      961 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     1021 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     1081 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     1141 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     1201 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     1261 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     1321 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     1381 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     1441 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     1501 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     1561 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     1621 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     1681 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     1741 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1801 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgggtcgac
     1861 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     1921 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     1981 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     2041 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     2101 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     2161 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     2221 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
     2281 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
     2341 gggcgggggg ggggggggsg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg
     2401 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
     2461 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
     2521 gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
     2581 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
     2641 tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg tgaaagcctt
     2701 gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt
     2761 gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg
     2821 cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt
     2881 gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg
     2941 ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct gcacccccct
     3001 ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg
     3061 gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg
     3121 cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc cggcggctgt
     3181 cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga
     3241 cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg caccccctct
     3301 agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     3361 cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg
     3421 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     3481 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     3541 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattgatggc agaaatcggt
     3601 actggctttc cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat gcactacgtc
     3661 gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc gacctcctcc
     3721 tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat tgctccagac
     3781 ctgatcggta tgggcaaatc cgacaaacca gacctgggtt atttcttcga cgaccacgtc
     3841 cgcttcatgg atgccttcat cgaagccctg ggtctggaag aggtcgtcct ggtcattcac
     3901 gactggggct ccgctctggg tttccactgg gccaagcgca atccagagcg cgtcaaaggt
     3961 attgcattta tggagttcat ccgccctatc ccgacctggg acgaatggcc agaatttgcc
     4021 cgcgagacct tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat catcgatcag
     4081 aacgttttta tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac tgaagtcgag
     4141 atggaccatt accgcgagcc gttcctgaat cctgttgacc gcgagccact gtggcgcttc
     4201 ccaaacgagc tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt cgaagaatac
     4261 atggactggc tgcaccagtc ccctgtcccg aagctgctgt tctggggcac cccaggcgtt
     4321 ctgatcccac cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg caaggctgtg
     4381 gacatcggcc cgggtctgaa tctgctgcaa gaagacaacc cggacctgat cggcagcgag
     4441 atcgcgcgct ggctgtcgac gctcgagatt tccggcggct ccggtgccag tgctggtggt
     4501 ggcagcctcg agatttccgg cgggaagaaa aagaagaaga agtccaagac aaaatgcgtg
     4561 attatgtagg cggccgccac cgcggtggag ctccagcttt tgttcccttt agtgagggtt
     4621 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4681 aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc
     4741 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga
     4801 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
     4861 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
     4921 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
     4981 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
     5041 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
     5101 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
     5161 tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc
     5221 agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5281 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5341 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5401 tcgtgccagc ggatccgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     5461 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     5521 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     5581 gaggcttttt tggaggccta ggcttttgca aaaagctaac ttgtttattg cagcttataa
     5641 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     5701 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccgctgcat
     5761 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5821 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5881 aaggcggt
//
