LOCUS       sequence-117436-        4374 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Middle entry nuclear localized tdTomato reporter with 6xMT and 2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4374)
  AUTHORS   Shin M, Beane TJ, Quillien A, Male I, Zhu LJ, Lawson ND
  TITLE     Vegfa signals through ERK to promote angiogenesis, but not artery 
            differentiation.
  JOURNAL   Development. 2016 Oct 15;143(20):3796-3805. Epub 2016 Aug 30.
  PUBMED    27578780
REFERENCE   2  (bases 1 to 4374)
  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: "2016-10-15"; volume: "143(20)"; pages: 
            "3796-3805. Epub 2016 Aug 30"
COMMENT     Sequence Label: pME-6MTnlstdTomato2A
FEATURES             Location/Qualifiers
     source          1..4374
                     /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      671..680
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             707..736
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             746..775
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             785..814
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             824..853
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             863..892
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             926..955
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             980..1000
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1001..2428
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     CDS             2441..2497
                     /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(2499..2598)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2615..2634)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2616..2634)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2639..2655)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2639..2655)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2757..2776)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2768..3577
                     /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(2835..2854)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3724..4312
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4213..4232
                     /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 agcaggctcc gccaccatgg ctggatccca tcgatttaaa gctatggagc aaaagctcat
      721 ttctgaagag gacttgaatg aaatggagca aaagctcatt tctgaagagg acttgaatga
      781 aatggagcaa aagctcattt ctgaagagga cttgaatgaa atggagcaaa agctcatttc
      841 tgaagaggac ttgaatgaaa tggagcaaaa gctcatttct gaagaggact tgaatgaaat
      901 ggagagcttg ggcgacctca ccatggagca aaagctcatt tctgaagagg acttgaattt
      961 agagatctcg aattccatgc ccaagaagaa gaggaaggtg atggtgagca agggcgagga
     1021 ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag ggctccatga acggccacga
     1081 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     1141 gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcat
     1201 gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc cccgattaca agaagctgtc
     1261 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gtctggtgac
     1321 cgtgacccag gactcctccc tgcaggacgg cacgctgatc tacaaggtga agatgcgcgg
     1381 caccaacttc ccccccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     1441 caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccacc aggccctgaa
     1501 gctgaaggac ggcggccact acctggtgga gttcaagacc atctacatgg ccaagaagcc
     1561 cgtgcaactg cccggctact actacgtgga caccaagctg gacatcacct cccacaacga
     1621 ggactacacc atcgtggaac agtacgagcg ctccgagggc cgccaccacc tgttcctggg
     1681 gcatggcacc ggcagcaccg gcagcggcag ctccggcacc gcctcctccg aggacaacaa
     1741 catggccgtc atcaaagagt tcatgcgctt caaggtgcgc atggagggct ccatgaacgg
     1801 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     1861 caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccccca
     1921 gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc gacatccccg attacaagaa
     1981 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggtct
     2041 ggtgaccgtg acccaggact cctccctgca ggacggcacg ctgatctaca aggtgaagat
     2101 gcgcggcacc aacttccccc ccgacggccc cgtaatgcag aagaagacca tgggctggga
     2161 ggcctccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga tccaccaggc
     2221 cctgaagctg aaggacggcg gccactacct ggtggagttc aagaccatct acatggccaa
     2281 gaagcccgtg caactgcccg gctactacta cgtggacacc aagctggaca tcacctccca
     2341 caacgaggac tacaccatcg tggaacagta cgagcgctcc gagggccgcc accacctgtt
     2401 cctgtacggc atggacgagc tgtacaaggg aggatccgga gccacgaact tctctctgtt
     2461 aaagcaagca ggagacgtgg aagaaaaccc cggtcctcac ccagctttct tgtacaaagt
     2521 tggcattata agaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca
     2581 aaataaaatc attatttgcc atccagctga tatcccctat agtgagtcgt attacatggt
     2641 catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca
     2701 agataaaata atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag
     2761 gggtgttatg agccatattc aacgggaaac gtcgaggccg cgattaaatt ccaacatgga
     2821 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     2881 ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     2941 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     3001 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     3061 gatccccgga aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     3121 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     3181 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     3241 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     3301 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     3361 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     3421 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     3481 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     3541 attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta
     3601 acactggcag agcattacgc tgacttgacg ggacggcgca agctcatgac caaaatccct
     3661 taacgtgagt tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3721 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3781 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3841 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     3901 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3961 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4021 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4081 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4141 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4201 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4261 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4321 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtt
//
