LOCUS       sequence-462711-        5006 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A middle entry gateway clone containing V5 tagged human LEF1 and an 
            H2A tagged mCherry, separated by a P2A peptide cleavage sequence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5006)
  AUTHORS   Cantu Gutierrez ME, Hill MC, Largoza G, Martin JF, Wythe JD
  TITLE     Defining the Transcriptional and Epigenetic Basis of Organotypic 
            Endothelial Diversity in the Developing and Adult Mouse
  JOURNAL   bioRxiv 2021.11.15.468651
REFERENCE   2  (bases 1 to 5006)
  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: "bioRxiv 
            2021.11.15.468651"
COMMENT     Sequence Label: JDW 1031 (pME_V5_LEF1_P2A_H2A_mCherry)
FEATURES             Location/Qualifiers
     source          1..5006
                     /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      675..684
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             684..725
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1950..2006
                     /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             2403..3113
                     /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(2548..2566)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2816..2835
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3010..3033
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(3131..3230)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3247..3266)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3248..3266)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3271..3287)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3271..3287)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3389..3408)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3400..4209
                     /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(3467..3486)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4356..4944
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4845..4864
                     /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 agcaggctgg atccgccacc atgggtaagc ctatccctaa ccctctcctc ggtctcgatt
      721 ctacgggtgg tgctggtggt atgccccaac tctccggagg aggtggcggc ggcggggggg
      781 acccggaact ctgcgccacg gacgagatga tccccttcaa ggacgagggc gatcctcaga
      841 aggaaaagat cttcgccgag atcagtcatc ccgaagagga aggcgattta gctgacatca
      901 agtcttcctt ggtgaacgag tctgaaatca tcccggccag caacggacac gaggtggcca
      961 gacaagcaca aacctctcag gagccctacc acgacaaggc cagagaacac cccgatgacg
     1021 gaaagcatcc agatggaggc ctctacaaca agggaccctc ctactcgagt tattccgggt
     1081 acataatgat gccaaatatg aataacgacc catacatgtc aaatggatct ctttctccac
     1141 ccatcccgag aacatcaaat aaagtgcccg tggtgcagcc atcccatgcg gtccatcctc
     1201 tcacccccct catcacttac agtgacgagc acttttctcc aggatcacac ccgtcacaca
     1261 tcccatcaga tgtcaactcc aaacaaggca tgtccagaca tcctccagct cctgatatcc
     1321 ctacttttta tcccttgtct ccgggtggtg ttggacagat caccccacct cttggctggc
     1381 aaggtcagcc tgtatatccc atcacgggtg gattcaggca accctaccca tcctcactgt
     1441 cagtcgacac ttccatgtcc aggttttccc atcatatgat tcccggtcct cctggtcccc
     1501 acacaactgg catccctcat ccagctattg taacacctca ggtcaaacag gaacatcccc
     1561 acactgacag tgacctaatg cacgtgaagc ctcagcatga acagagaaag gagcaggagc
     1621 caaaaagacc tcacattaag aagcctctga atgcttttat gttatacatg aaagaaatga
     1681 gagcgaatgt cgttgctgag tgtactctaa aagaaagtgc agctatcaac cagattcttg
     1741 gcagaaggtg gcatgccctc tcccgtgaag agcaggctaa atattatgaa ttagcacgga
     1801 aagaaagaca gctacatatg cagctttatc caggctggtc tgcaagagac aattatggta
     1861 agaaaaagaa gaggaagaga gagaaactac aggaatctgc atcaggtaca ggtccaagaa
     1921 tgacagctgc ctacatctgc ggtaccggag ccactaactt ctccctgttg aaacaagcag
     1981 gggatgtcga agagaatccc gggccaccta ggatggcagg tggaaaagca ggtaaagaca
     2041 gtggaaaagc caagacgaaa gccgtctcca gatctcagag agctggtctg cagttccctg
     2101 taggtcgtat ccacaggcac ctgaagacac gaaccaccag ccatggccgt gtcggagcta
     2161 cagctgcggt ttacagtgct gctatcctgg agtatctcac cgctgaggtg ctggagttgg
     2221 ctggaaacgc ttctaaggat ctgaaggtga agcgaatcac tcccagacat ttacagctgg
     2281 ccatccgcgg agacgaggag ctggactcgc ttatcaaggc caccattgct ggaggaggag
     2341 tcattcctca catccacaag tctctgattg gtaagaaggg ccagcagaaa accgcagggg
     2401 ggatggtgag caagggcgag gaggacaaca tggccatcat caaggagttc atgcgcttca
     2461 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     2521 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc
     2581 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
     2641 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
     2701 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
     2761 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     2821 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     2881 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     2941 ccgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
     3001 tcaacatcaa gctggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     3061 agcgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag taatgatagc
     3121 tcgagagatc tcccagcttt cttgtacaaa gttggcatta taagaaagca ttgcttatca
     3181 atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct
     3241 gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc
     3301 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     3361 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     3421 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     3481 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
     3541 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     3601 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     3661 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta
     3721 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     3781 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     3841 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     3901 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     3961 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     4021 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     4081 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     4141 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     4201 tttttctaat cagaattggt taattggttg taacactggc agagcattac gctgacttga
     4261 cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     4321 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4381 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4441 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4501 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4561 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     4621 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     4681 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     4741 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4801 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4861 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4921 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4981 ttttgctggc cttttgctca catgtt
//
