LOCUS       sequence-447361-        5091 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway compatible middle entry clone containing 
            GalT-mTagBFP-HA-P2A-H2A-iRFP (BFP golgi reporter and infrared RFP 
            nuclear reporter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5091)
  AUTHORS   Gillespie WB 3rd, Zhang Y, Ruiz OE, Cerda J 3rd, Ortiz-Guzman J, 
            Sherman M, Turner WD, Largoza G, Mosser LE, Fujimoto E, Chien CB, 
            Kwan KM, Arenkiel BR, Devine WP, Wythe JD
  TITLE     MultiSite Assembly of Gateway Induced Clones (MAGIC): a flexible 
            cloning toolbox for use in vertebrate model systems.
  JOURNAL   Development. 2025 Nov 15;152(22):dev204308. doi: 10.1242/dev.204308.
            Epub 2025 Nov 13.
  PUBMED    41230856
REFERENCE   2  (bases 1 to 5091)
  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"; journalName: 
            "Development."; date: "2025-11-15"; volume: "152(22):dev204308. 
            doi"; pages: " 10.1242/dev.204308. Epub 2025 Nov 13"
COMMENT     Sequence Label: pME_Golgi-BFP_P2A_H2A_iRFP (JDW 1007)
FEATURES             Location/Qualifiers
     source          1..5091
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             970..1668
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             1669..1695
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             1714..1770
                     /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"
     protein_bind    complement(3216..3315)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3332..3351)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3333..3351)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3356..3372)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3356..3372)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3474..3493)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3485..4294
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3552..3571)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4441..5029
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4930..4949
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc ttaattaaga attctctaga gccaccatga ggcttcggga gccgctcctg
      721 agcggcagcg ccgcgatgcc aggcgcgtcc ctacagcggg cctgccgcct gctcgtggcc
      781 gtctgcgctc tgcaccttgg cgtcaccctc gtttactacc tggctggccg cgacctgagc
      841 cgcctgcccc aactggtcgg agtctccaca ccgctgcagg gcggctcgaa cagtgccgcc
      901 gccatcgggc agtcctccgg ggagctccgg accggagggg ccaaggatcc acccgtcgcc
      961 accatcgata tgagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc
     1021 accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
     1081 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
     1141 ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc
     1201 gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa
     1261 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
     1321 aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca
     1381 ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga
     1441 aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacgc caagaccaca
     1501 tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac
     1561 agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca
     1621 gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaatta tgacgtccca
     1681 gattacgcaa gtttgcccag atctggcagc ggcgccacga acttctctct gttaaagcaa
     1741 gcaggcgacg tggaagaaaa ccccggtccc ggtaccatgg caggtggaaa agcaggtaaa
     1801 gacagtggaa aagccaagac gaaagccgtc tccaggtctc agagagctgg tctgcagttc
     1861 cctgtaggtc gtatccacag gcacctgaag acccgaacca ccagccatgg ccgtgtcgga
     1921 gctacagctg cggtttacag tgctgctatc ctggagtatc tcaccgctga ggtgctggag
     1981 ttggctggaa acgcttctaa ggatctgaag gtgaagcgaa tcactcccag acatttacag
     2041 ctggccatcc gcggagacga ggagctggac tcgcttatca aggccaccat tgctggagga
     2101 ggagtcattc ctcacatcca caagtctctg attggtaaga agggccagca gaaaaccgca
     2161 gatccaccgg tcgacaccat ggccgaaggg agcgtggcaa gacagcccga tctgctgaca
     2221 tgcgacgacg aaccaatcca cattccagga gctatccagc ctcatggact gctgctggct
     2281 ctggccgctg acatgactat cgtggcaggc tctgataacc tgccagagct gaccgggctg
     2341 gctatcggag cactgattgg ccggtccgca gccgacgtgt tcgattctga gactcacaat
     2401 agactgacca tcgccctggc tgaacctggg gctgcagtgg gagccccaat taccgtcggc
     2461 ttcacaatgc gaaaggacgc tggctttatc gggtcctggc accgccatga tcagctgatt
     2521 tttctggagc tggaaccacc tcagagagac gtggcagagc cacaggcttt ctttcggaga
     2581 acaaacagcg ccatcaggcg cctgcaggcc gctgagactc tggaatccgc ttgcgcagcc
     2641 gctgcacagg aagtgaggaa gatcaccggg ttcgatcgcg tcatgatcta cagattcgcc
     2701 tcagacttta gcggagaagt gatcgcagaa gatcgctgtg ccgaggtcga aagtaaactg
     2761 ggcctgcatt acccagcctc aaccgtgcct gcacaggcac gacggctgta tacaatcaac
     2821 cccgtgcgga tcattcccga cattaattac agaccagtgc ccgtcacacc tgacctgaac
     2881 ccagtgactg ggaggcccat tgatctgagc ttcgccattc tgcgcagcgt gtcccctgtc
     2941 cacctggagt ttatgaggaa tatcggaatg catggcacca tgtctatcag tattctgaga
     3001 ggcgaaaggc tgtgggggct gatcgtgtgc caccatcgga caccttacta tgtggacctg
     3061 gacggcaggc aggcttgtga actggtggct caggtgctgg cttggcagat tggcgtgatg
     3121 aaggaggggc atggcaccgg cagcaccggc agcggcagca gcggccgctc cggcctcgag
     3181 taatgataaa ctagtgttta aactacgtaa ctaggaccca gctttcttgt acaaagttgg
     3241 cattataaga aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa
     3301 taaaatcatt atttgccatc cagctgatat cccctatagt gagtcgtatt acatggtcat
     3361 agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga
     3421 taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     3481 tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc
     3541 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     3601 tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     3661 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     3721 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     3781 ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     3841 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     3901 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     3961 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     4021 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     4081 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     4141 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     4201 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     4261 gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca
     4321 ctggcagagc attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa
     4381 cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4441 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4501 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4561 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4621 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4681 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4741 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4801 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4861 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4921 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4981 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5041 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt t
//
