LOCUS       sequence-398026-        5602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HaloTag-eGFP tandem (cytosolic).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5602)
  AUTHORS   Ebner M, Lucic I, Leonard TA, Yudushkin I
  TITLE     PI(3,4,5)P3 Engagement Restricts Akt Activity to Cellular Membranes.
  JOURNAL   Mol Cell. 2017 Feb 2;65(3):416-431.e6. doi: 
            10.1016/j.molcel.2016.12.028.
  PUBMED    28157504
REFERENCE   2  (bases 1 to 5602)
  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: 
            "10.1016/j.molcel.2016.12"; journalName: "Mol Cell"; date: 
            "2017-02-2- 2"; volume: "65"; issue: "3"; pages: "416-431.e6"
COMMENT     Sequence Label: pHaloTag-EGFP
FEATURES             Location/Qualifiers
     source          1..5602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        21..212
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     169..189
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(214..318)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      345..800
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(432..451)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     642..663
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    807..928
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(818..837)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     872..891
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(1051..1764,1765..1767))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(1054..1764,1765..1767))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1096..1117)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1445..1464
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1704..1725
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1822..2712)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     regulatory      2709..2718
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2750..2953)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2779..2799)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2954..3257
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(3432..4020)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3512..3531)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4349..4396
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4421..4440
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4628..5422)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(4628..5422)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4739..4758)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5349..5368
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(5457..5602,1..212))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(5471..5602,1..4)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca
       61 tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac taattgagat
      121 gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac
      181 acacattcca cagctggttc tttccgcctc aggactcttc ctttttcaat attattgaag
      241 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
      301 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgcgc cctgtagcgg
      361 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
      421 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
      481 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
      541 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
      601 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
      661 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
      721 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
      781 aatattaacg cttacaattt acgccttaag atacattgat gagtttggac aaaccacaac
      841 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
      901 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
      961 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat
     1021 ggctgattat gatctagagt cgcggccgct ttacttgtac agctcgtcca tgccgagagt
     1081 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc
     1141 tttgctcagg gcggactggg tgctcaggta gtggttgtcg ggcagcagca cggggccgtc
     1201 gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt
     1261 gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac
     1321 gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa
     1381 gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc
     1441 gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc
     1501 gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca
     1561 ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt
     1621 ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga
     1681 cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc
     1741 ggtgaacagc tcctcgccct tgctcaccaa ggttgcgacc ggtggatccc gggcccgcgg
     1801 tacccaaggt tgcgaccggt ggccggaaat ctcgagcgtc gacagccagc gcgcgatctc
     1861 gctgccgatc aggtccgggt tgtcttcttg cagcagattc agacccgggc cgatgtccac
     1921 agccttgcag ttaggcaggc ttttggccag gcgagcggct tcggccggtg ggatcagaac
     1981 gcctggggtg ccccagaaca gcagcttcgg gacaggggac tggtgcagcc agtccatgta
     2041 ttcttcgacc agcgcgacga tgttcgctgg ctcaccggcg attggcagct cgtttgggaa
     2101 gcgccacagt ggctcgcggt caacaggatt caggaacggc tcgcggtaat ggtccatctc
     2161 gacttcagtc agcgggcgga cgacacccat cggcagcgta ccctcgataa aaacgttctg
     2221 atcgatgatc agcttgcggc cgacgtcggt ggtgcggaag gcctggaagg tctcgcgggc
     2281 aaattctggc cattcgtccc aggtcgggat agggcggatg aactccataa atgcaatacc
     2341 tttgacgcgc tctggattgc gcttggccca gtggaaaccc agagcggagc cccagtcgtg
     2401 aatgaccagg acgacctctt ccagacccag ggcttcgatg aaggcatcca tgaagcggac
     2461 gtggtcgtcg aagaaataac ccaggtctgg tttgtcggat ttgcccatac cgatcaggtc
     2521 tggagcaatg cagcgatggg tcggtgcaac atgcgggatg atgttgcgcc acacgtagga
     2581 ggaggtcggg ttaccgtgca ggaacagcac aggggtgcca tcgcgcggac caacatcgac
     2641 gtagtgcatg cgctcgccca ggacttccac ataatggggg tcgaatggaa agccagtacc
     2701 gatttctgcc atggtggctt tgctagcgga tctgacggtt cactaaacca gctctgctta
     2761 tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac
     2821 gacattttgg aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg
     2881 ggtggagact tggaaatccc cgtgagtcaa accgctatcc acgcccattg atgtactgcc
     2941 aaaaccgcat caccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa
     3001 agtcccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt
     3061 caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc
     3121 gtaaatactc cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat
     3181 acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta
     3241 ccgtaagtta tgtaacgcgg aactccatat atgggctatg aactaatgac cccgtaattg
     3301 attactatta ataactaatg catggcggta atacggttat ccacagaatc aggggataac
     3361 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3421 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3481 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3541 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3601 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3661 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3721 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3781 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3841 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3901 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3961 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4021 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4081 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4141 tgaagtttta aatcaatcta aagtatatat gagtaacctg aggctatggc agggcctgcc
     4201 gccccgacgt tggctgcgag ccctgggcct tcacccgaac ttggggggtg gggtggggaa
     4261 aaggaagaaa cgcgggcgta ttggccccaa tggggtctcg gtggggtatc gacagagtgc
     4321 cagccctggg accgaacccc gcgtttatga acaaacgacc caacaccgtg cgttttattc
     4381 tgtcttttta ttgccgtcat agcgcgggtt ccttccggta ttgtctcctt ccgtgtttca
     4441 gttagcctcc ccctagggtg ggcgaagaac tccagcatga gatccccgcg ctggaggatc
     4501 atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa ggcggcggtg
     4561 gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc gaaccccaga
     4621 gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag
     4681 cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa
     4741 tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt
     4801 cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat
     4861 gggtcacgac gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg
     4921 ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca
     4981 tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg
     5041 gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag
     5101 caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc
     5161 ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg
     5221 atagccgcgc tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa
     5281 aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg
     5341 tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt
     5401 gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga tcgatctttg
     5461 caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc agaggccgag
     5521 gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg
     5581 gaactgggcg gagttagggg cg
//
