LOCUS       sequence-446939-        4491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible middle entry clone containing Biotin Ligase 
            followed by T2A and mCherry membrane reporter (biotinylation and 
            flourescent cell membrane reporter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4491)
  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 4491)
  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-3xHA-BirA-T2A-mCherry-caax (JDW 889)
FEATURES             Location/Qualifiers
     source          1..4491
                     /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"
     regulatory      669..678
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             684..710
                     /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             717..743
                     /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             750..776
                     /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             792..1754
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             792..1754
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1770..1823
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1830..2537
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1975..1993)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2243..2262
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2616..2715)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2732..2751)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2733..2751)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2756..2772)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2756..2772)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2874..2893)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2885..3694
                     /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(2952..2971)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3841..4429
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4330..4349
                     /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 caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctgc caccatggcc acctatgacg tcccagatta cgcaagtttg cccgggtatg
      721 atgttcctga ttatgctagc ctcccgggtt acgatgtgcc cgactatgcc tcacttccag
      781 gcagatctcc tatgaaggat aacaccgtgc cactgaaatt gattgccctg ttagcgaacg
      841 gtgaatttca ctctggcgag cagttgggtg aaacgctggg aatgagccgg gcggctatta
      901 ataaacacat tcagacactg cgtgactggg gcgttgatgt ctttaccgtt ccgggtaaag
      961 gatacagcct gcctgagcct atccagttac ttaatgctaa acagatattg ggtcagctgg
     1021 atggcggtag tgtagccgtg ctgccagtga ttgactccac gaatcagtac cttcttgatc
     1081 gtatcggaga gcttaaatcg ggcgatgctt gcattgcaga ataccagcag gctggccgtg
     1141 gtcgccgggg tcggaaatgg ttttcgcctt ttggcgcaaa cttatatttg tcgatgttct
     1201 ggcgtctgga acaaggcccg gcggcggcga ttggtttaag tctggttatc ggtatcgtga
     1261 tggcggaagt attacgcaag ctgggtgcag ataaagttcg tgttaaatgg cctaatgacc
     1321 tctatctgca ggatcgcaag ctggcaggca ttctggtgga gctgactggc aaaactggcg
     1381 atgcggcgca aatagtcatt ggagccggga tcaacatggc aatgcgccgt gttgaagaga
     1441 gtgtcgttaa tcaggggtgg atcacgctgc aggaagcggg gatcaatctc gatcgtaata
     1501 cgttggcggc catgctaata cgtgaattac gtgctgcgtt ggaactcttc gaacaagaag
     1561 gattggcacc ttatctgtcg cgctgggaaa agctggataa ttttattaat cgcccagtga
     1621 aacttatcat tggtgataaa gaaatatttg gcatttcacg cggaatagac aaacaggggg
     1681 ctttattact tgagcaggat ggaataataa aaccctggat gggcggtgaa atatccctgc
     1741 gtagtgcaga aaaagatatc ggatcaggag agggcagggg aagtcttcta acatgcgggg
     1801 acgtggagga aaatcccggg cccagatcta tggtgagcaa gggcgaggag gataacatgg
     1861 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg
     1921 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     1981 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca
     2041 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
     2101 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     2161 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
     2221 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct
     2281 cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga
     2341 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
     2401 ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg
     2461 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca
     2521 tggacgagct gtacaaggga ggaggaagat ctaagctgaa ccctcctgat gagagtggcc
     2581 ccggctgcat gagctgcaag tgtgtgctct cctgaaccca gctttcttgt acaaagttgg
     2641 cattataaga aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa
     2701 taaaatcatt atttgccatc cagctgatat cccctatagt gagtcgtatt acatggtcat
     2761 agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga
     2821 taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     2881 tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc
     2941 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     3001 tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     3061 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     3121 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     3181 ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     3241 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     3301 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     3361 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     3421 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     3481 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     3541 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     3601 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     3661 gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca
     3721 ctggcagagc attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa
     3781 cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3841 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3901 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3961 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4021 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4081 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4141 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4201 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4261 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4321 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4381 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4441 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt t
//
