LOCUS       sequence-308104-        7333 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express APEX2-Galectin-8 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7333)
  AUTHORS   Jia J, Abudu YP, Claude-Taupin A, Gu Y, Kumar S, Choi SW, Peters R, 
            Mudd MH, Allers L, Salemi M, Phinney B, Johansen T, Deretic V
  TITLE     Galectins Control mTOR in Response to Endomembrane Damage.
  JOURNAL   Mol Cell. 2018 Apr 5;70(1):120-135.e8. doi: 
            10.1016/j.molcel.2018.03.009.
  PUBMED    29625033
REFERENCE   2  (bases 1 to 7333)
  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.2018.03"; journalName: "Mol Cell"; date: 
            "2018-04-5- 5"; volume: "70"; issue: "1"; pages: "120-135.e8"
COMMENT     Sequence Label: pJJiaDEST-APEX2-GAL8
FEATURES             Location/Qualifiers
     source          1..7333
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             910..933
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             934..1680
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     CDS             1687..1713
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     protein_bind    1726..1750
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2831..2855)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2886..2904)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2887..2904)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2924..2941)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2930..3154
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3200..3628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3287..3306)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3497..3518
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3637..3657)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3642..3971
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3822..3957
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3884..3903
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4038..4832
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4092..4111)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4702..4721
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5006..5127
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5043..5062)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5097..5116
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5176..5192)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5176..5192)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5189..5211)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5200..5216
                     /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(5224..5230,5231..5248,5249..5254))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5269..5290
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5407..5424)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5578..6166)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5658..5677)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6337..7128,7129..7197))
                     /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     6960..6979
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7198..7302)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg actacaagga tgacgacgat aagggaaagt cttacccaac tgtgagtgct
      961 gattaccagg acgccgttga gaaggcgaag aagaagctca gaggcttcat cgctgagaag
     1021 agatgcgctc ctctaatgct ccgtttggca ttccactctg ctggaacctt tgacaagggc
     1081 acgaagaccg gtggaccctt cggaaccatc aagcaccctg ccgaactggc tcacagcgct
     1141 aacaacggtc ttgacatcgc tgttaggctt ttggagccac tcaaggcgga gttccctatt
     1201 ttgagctacg ccgatttcta ccagttggct ggcgttgttg ccgttgaggt cacgggtgga
     1261 cctaaggttc cattccaccc tggaagagag gacaagcctg agccaccacc agagggtcgc
     1321 ttgcccgatc ccactaaggg ttctgaccat ttgagagatg tgtttggcaa agctatgggg
     1381 cttactgacc aagatatcgt tgctctatct gggggtcaca ctattggagc tgcacacaag
     1441 gagcgttctg gatttgaggg tccctggacc tctaatcctc ttattttcga caactcatac
     1501 ttcacggagt tgttgagtgg tgagaaggaa ggtctccttc agctaccttc tgacaaggct
     1561 cttttgtctg accctgtatt ccgccctctc gttgacaaat atgcagcgga cgaagatgcc
     1621 ttctttgctg attacgctga ggctcaccaa aagctttccg agcttgggtt tgctgatgcc
     1681 ctgcagctgc ctcccctgga gcgcctgacc ctggacctcg aatcaacaag tttgtacaaa
     1741 aaagcaggct tcttgtcctt aaacaaccta cagaatatca tctataaccc ggtaatcccg
     1801 tttgttggca ccattcctga tcagctggat cctggaactt tgattgtgat acgtgggcat
     1861 gttcctagtg acgcagacag attccaggtg gatctgcaga atggcagcag catgaaacct
     1921 cgagccgatg tggcctttca tttcaatcct cgtttcaaaa gggccggctg cattgtttgc
     1981 aatactttga taaatgaaaa atggggacgg gaagagatca cctatgacac gcctttcaaa
     2041 agagaaaagt cttttgagat cgtgattatg gtgctgaagg acaaattcca ggtggctgta
     2101 aatggaaaac atactctgct ctatggccac aggatcggcc cagagaaaat agacactctg
     2161 ggcatttatg gcaaagtgaa tattcactca attggtttta gcttcagctc ggacttacaa
     2221 agtacccaag catctagtct ggaactgaca gagataagta gagaaaatgt tccaaagtct
     2281 ggcacgcccc agcttcctag taatagagga ggagacattt ctaaaatcgc acccagaact
     2341 gtctacacca agagcaaaga ttcgactgtc aatcacactt tgacttgcac caaaatacca
     2401 cctatgaact atgtgtcaaa gaggctgcca ttcgctgcaa ggttgaacac ccccatgggc
     2461 cctggacgaa ctgtcgtcgt taaaggagaa gtgaatgcaa atgccaaaag ctttaatgtt
     2521 gacctactag caggaaaatc aaaggatatt gctctacact tgaacccacg cctgaatatt
     2581 aaagcatttg taagaaattc ttttcttcag gagtcctggg gagaagaaga gagaaatatt
     2641 acctctttcc catttagtcc tgggatgtac tttgagatga taatttattg tgatgttaga
     2701 gaattcaagg ttgcagtaaa tggcgtacac agcctggagt acaaacacag atttaaagag
     2761 ctcagcagta ttgacacgct ggaaattaat ggagacatcc acttactgga agtaaggagc
     2821 tggtagtagg acccagcttt cttgtacaaa gtggttgatt cgagcatgca tctagagggc
     2881 cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac tgtgccttct
     2941 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3001 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3061 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     3121 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     3181 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3241 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3301 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3361 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3421 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3481 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3541 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3601 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     3661 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3721 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3781 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3841 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3901 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3961 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     4021 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     4081 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     4141 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     4201 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     4261 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     4321 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     4381 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     4441 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4501 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4561 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4621 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4681 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4741 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4801 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     4861 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     4921 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4981 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     5041 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     5101 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     5161 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     5221 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5281 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5341 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5401 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5461 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5521 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5581 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5641 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5701 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5761 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5821 gctgggctgt gtgcacaaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5881 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5941 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6001 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6061 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6121 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6181 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6241 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6301 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6361 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6421 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6481 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6541 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6601 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6661 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6721 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6781 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6841 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6901 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6961 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7021 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7081 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7141 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7201 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7261 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7321 gccacctgac gtc
//
