LOCUS       sequence-308098-        7042 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express APEX2-Galectin-3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7042)
  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 7042)
  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-GAL3
FEATURES             Location/Qualifiers
     source          1..7042
                     /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(2540..2564)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2595..2613)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2596..2613)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2633..2650)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2639..2863
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2909..3337
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2996..3015)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3206..3227
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3346..3366)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3351..3680
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3531..3666
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3593..3612
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3747..4541
                     /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(3801..3820)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4411..4430
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4715..4836
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4752..4771)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4806..4825
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4885..4901)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4885..4901)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4898..4920)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4909..4925
                     /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(4933..4939,4940..4957,4958..4963))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4978..4999
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5116..5133)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5287..5875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5367..5386)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6046..6837,6838..6906))
                     /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     6669..6688
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6907..7011)
                     /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 ccgcggccgc ccccttcacc atggcagaca atttttcgct ccatgatgcg
     1801 ttatctgggt ctggaaaccc aaaccctcaa ggatggcctg gcgcatgggg gaaccagcct
     1861 gctggggcag ggggctaccc aggggcttcc tatcctgggg cctaccccgg gcaggcaccc
     1921 ccaggggctt atcctggaca ggcacctcca ggcgcctacc ctggagcacc tggagcttat
     1981 cccggagcac ctgcacctgg agtctaccca gggccaccca gcggccctgg ggcctaccca
     2041 tcttctggac agccaagtgc caccggagcc taccctgcca ctggccccta tggcgcccct
     2101 gctgggccac tgattgtgcc ttataacctg cctttgcctg ggggagtggt gcctcgcatg
     2161 ctgataacaa ttctgggcac ggtgaagccc aatgcaaaca gaattgcttt agatttccaa
     2221 agagggaatg atgttgcctt ccactttaac ccacgcttca atgagaacaa caggagagtc
     2281 attgtttgca atacaaagct ggataataac tggggaaggg aagaaagaca gtcggttttc
     2341 ccatttgaaa gtgggaaacc attcaaaata caagtactgg ttgaacctga ccacttcaag
     2401 gttgcagtga atgatgctca cttgttgcag tacaatcatc gggttaaaaa actcaatgaa
     2461 atcagcaaac tgggaatttc tggtgacata gacctcacca gtgcttcata taccatgata
     2521 taaaagggtg ggcgcgccga cccagctttc ttgtacaaag tggttgattc gagcatgcat
     2581 ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc agcctcgact
     2641 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2701 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     2761 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2821 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     2881 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     2941 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3001 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3061 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3121 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3181 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3241 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     3301 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     3361 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3421 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3481 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     3541 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     3601 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     3661 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     3721 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3781 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3841 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3901 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     3961 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     4021 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     4081 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     4141 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4201 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4261 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4321 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4381 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4441 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     4501 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     4561 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     4621 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4681 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     4741 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4801 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     4861 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4921 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4981 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5041 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5101 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5161 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5221 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5281 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5341 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5401 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5461 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5521 tcgctccaag ctgggctgtg tgcacaaacc ccccgttcag cccgaccgct gcgccttatc
     5581 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5641 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5701 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5761 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5821 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5881 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5941 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6001 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6061 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6121 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6181 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6241 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6301 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6361 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6421 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6481 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6541 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6601 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6661 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6721 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6781 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6841 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6901 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6961 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     7021 ccgaaaagtg ccacctgacg tc
//
