LOCUS       Exported                6228 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Mammalian expression vector driving APEX2-2xFYVE (2xMouse FYVE 
            domain from HRS, [marks PI3P] fused to EM peroxidase marker). Also 
            contains bicistronially expressed cytoplasmic red reporter..
ACCESSION   .
VERSION     .
KEYWORDS    mKate2-P2A-APEX2-2xFYVE_hrs
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6228)
  AUTHORS   Ariotti N, Hall TE, Rae J, Ferguson C, McMahon KA, Martel N, Webb 
            RE, Webb RI, Teasdale RD, Parton RG
  TITLE     Modular Detection of GFP-Labeled Proteins for Rapid Screening by 
            Electron Microscopy in Cells and Organisms.
  JOURNAL   Dev Cell. 2015 Nov 23;35(4):513-25. doi: 
            10.1016/j.devcel.2015.10.016. Epub 2015 Nov 12.
  PUBMED    26585296
REFERENCE   2  (bases 1 to 6228)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6228
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    108..132
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      133..142
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             139..834
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     CDS             850..906
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             925..1671
                     /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"
     protein_bind    complement(1676..1700)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2217..2237)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        complement(2255..2272)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2277..2411
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2301..2320)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2355..2374
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2524..2544)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2524..2542)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2563..2579)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2563..2579)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2576..2598)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2587..2603
                     /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(2611..2641)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2656..2677
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2794..2811)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2965..3553)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3045..3064)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3724..4584)
                     /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     4347..4366
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4585..4689)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4715..5170)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4852..4873)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5064..5083
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5244..6228
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaattca aggatcaaca agtttgtaca
      121 aaaaagcagg ctgccaccat ggtgagcgag ctgattaagg agaacatgca catgaagctg
      181 tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag
      241 ccctacgagg gcacccagac catgagaatc aaggcggtcg agggcggccc tctccccttc
      301 gccttcgaca tcctggctac cagcttcatg tacggcagca aaaccttcat caaccacacc
      361 cagggcatcc ccgacttctt taagcagtcc ttccccgagg gcttcacatg ggagagagtc
      421 accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc
      481 tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg
      541 cagaagaaaa cactcggctg ggaggcctcc accgagaccc tgtaccccgc tgacggcggc
      601 ctggaaggca gagccgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac
      661 ttgaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac
      721 tatgtggaca gaagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag
      781 cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca cagacctagg
      841 ggcagcggcg ccaccaactt cagcctgctg aagcaggccg gcgacgtgga ggagaacccc
      901 ggccccgccc ccggcagcct cgagggaaag tcttacccaa ctgtgagtgc tgattaccag
      961 gacgccgttg agaaggcgaa gaagaagctc agaggcttca tcgctgagaa gagatgcgct
     1021 cctctaatgc tccgtttggc attccactct gctggaacct ttgacaaggg cacgaagacc
     1081 ggtggaccct tcggaaccat caagcaccct gccgaactgg ctcacagcgc taacaacggt
     1141 cttgacatcg ctgttaggct tttggagcca ctcaaggcgg agttccctat tttgagctac
     1201 gccgatttct accagttggc tggcgttgtt gccgttgagg tcacgggtgg acctaaggtt
     1261 ccattccacc ctggaagaga ggacaagcct gagccaccac cagagggtcg cttgcccgat
     1321 cccactaagg gttctgacca tttgagagat gtgtttggca aagctatggg gcttactgac
     1381 caagatatcg ttgctctatc tgggggtcac actattggag ctgcacacaa ggagcgttct
     1441 ggatttgagg gtccctggac ctctaatcct cttattttcg acaactcata cttcacggag
     1501 ttgttgagtg gtgagaagga aggtctcctt cagctacctt ctgacaaggc tcttttgtct
     1561 gaccctgtat tccgccctct cgttgacaaa tatgcagcgg acgaagatgc cttctttgct
     1621 gattacgctg aggctcacca aaagctttcc gagcttgggt ttgctgatgc cgtcgaccca
     1681 gctttcttgt acaaagtggc tctcgagctc aagcttcgaa ttgaattcga aagtgatgcc
     1741 atgttcgctg ctgaaagagc ccctgactgg gtggatgctg aggaatgcca tcggtgcaga
     1801 gtacagtttg gggtggtgac ccgcaagcat cactgccgag catgtgggca gatcttctgt
     1861 ggcaagtgct cctccaagta ctccaccatc cccaagttcg gcattgagaa ggaggtgcgc
     1921 gtgtgtgagc cctgctatga gcagctgaac aagaaggcac aaggacaagg atccgaaagt
     1981 gatgccatgt tcgctgctga aagagcccct gactgggtgg atgctgagga atgccatcgg
     2041 tgcagagtac agtttggggt ggtgacccgc aagcatcact gccgagcatg tgggcagatc
     2101 ttctgtggca agtgctcctc caagtactcc accatcccca agttcggcat tgagaaggag
     2161 gtgcgcgtgt gtgagccctg ctatgagcag ctgaacaaga aggcataagt cgacggcaac
     2221 tttattatac atagttgcac tcgagcctct agaactatag tgagtcgtat tacgtagatc
     2281 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     2341 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     2401 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
     2461 gggaggtttt ttaattcgcg gccgcggcgc caatgcattg ggcccggtac ccagcttttg
     2521 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
     2581 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     2641 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     2701 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     2761 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     2821 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     2881 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     2941 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3001 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3061 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3121 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3181 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3241 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3301 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3361 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     3421 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3481 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3541 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3601 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     3661 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3721 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3781 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3841 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     3901 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3961 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4021 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4081 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4141 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4201 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4261 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4321 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4381 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4441 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4501 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4561 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     4621 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4681 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta
     4741 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     4801 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     4861 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     4921 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     4981 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     5041 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     5101 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     5161 cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     5221 gcctcttcgc tattacgcca gtcgaccata gccaattcaa tatggcgtat atggactcat
     5281 gccaattcaa tatggtggat ctggacctgt gccaattcaa tatggcgtat atggactcgt
     5341 gccaattcaa tatggtggat ctggacccca gccaattcaa tatggcggac ttggcaccat
     5401 gccaattcaa tatggcggac ttggcactgt gccaactggg gaggggtcta cttggcacgg
     5461 tgccaagttt gaggaggggt cttggccctg tgccaagtcc gccatattga attggcatgg
     5521 tgccaataat ggcggccata ttggctatat gccaggatca atatataggc aatatccaat
     5581 atggccctat gccaatatgg ctattggcca ggttcaatac tatgtattgg ccctatgcca
     5641 tatagtattc catatatggg ttttcctatt gacgtagata gcccctccca atgggcggtc
     5701 ccatatacca tatatggggc ttcctaatac cgcccatagc cactccccca ttgacgtcaa
     5761 tggtctctat atatggtctt tcctattgac gtcatatggg cggtcctatt gacgtatatg
     5821 gcgcctcccc cattgacgtc aattacggta aatggcccgc ctggctcaat gcccattgac
     5881 gtcaatagga ccacccacca ttgacgtcaa tgggatggct cattgcccat tcatatccgt
     5941 tctcacgccc cctattgacg tcaatgacgg taaatggccc acttggcagt acatcaatat
     6001 ctattaatag taacttggca agtacattac tattggaagg acgccagggt acattggcag
     6061 tactcccatt gacgtcaatg gcggtaaatg gcccgcgatg gctgccaagt acatccccat
     6121 tgacgtcaat ggggaggggc aatgacgcaa atgggcgttc cattgacgta aatgggcggt
     6181 aggcgtgcct aatgggaggt ctatataagc aatgctcgtt tagggaac
//
