LOCUS       sequence-462713-        4961 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A middle entry gateway clone containing V5 tagged human TCF7 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 4961)
  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 4961)
  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 1034 (pME_V5_TCF7_P2A_H2A_mCherry)
FEATURES             Location/Qualifiers
     source          1..4961
                     /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             1905..1961
                     /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             2358..3068
                     /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(2503..2521)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2771..2790
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2965..2988
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(3086..3185)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3202..3221)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3203..3221)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3226..3242)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3226..3242)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3344..3363)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3355..4164
                     /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(3422..3441)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4311..4899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4800..4819
                     /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 atctgccacc atgggtaagc ctatccctaa ccctctcctc ggtctcgatt
      721 ctacgggtgg tgctggtggt atgccgcagc tggactccgg cgggggcggc gcgggcggcg
      781 gcgacgacct cggcgcgccg gacgagctgc tggccttcca ggatgaaggc gaggagcagg
      841 acgacaagag ccgcgacagc gccgccggtc ccgagcgcga cctggccgag ctcaagtcgt
      901 cgctcgtgaa cgagtccgag ggcgcggccg gcggcgcagg gatcccgggg gtcccggggg
      961 ccggcgccgg ggcccgcggc gaggccgagg ctctcgggcg ggaacacgct gcgcagagac
     1021 tcttcccgga caaacttcca gagcccctgg aggacggcct gaaggccccg gagtgcacca
     1081 gcggcatgta caaagagacc gtctactccg ccttcaatct gctcatgcat tacccacccc
     1141 cctcgggagc agggcagcac ccccagccgc agcccccgct gcacaaggcc aatcagcccc
     1201 cccacggtgt cccccaactc tctctctacg aacatttcaa cagcccacat cccacccctg
     1261 cacctgcgga catcagccag aagcaagttc acaggcctct gcagacccct gacctctctg
     1321 gcttctactc cctgacctca ggcagcatgg ggcagctccc ccacactgtg agctggttca
     1381 cccacccatc cttgatgcta ggttctggtg tacctggtca cccagcagcc atcccccacc
     1441 cggccattgt gcccccctca gggaagcagg agctgcagcc cttcgaccgc aacctgaaga
     1501 cacaagcaga gtccaaggca gagaaggagg ccaagaagcc aaccatcaag aagcccctca
     1561 atgccttcat gctgtacatg aaggagatga gagccaaggt cattgcagag tgcacactta
     1621 aggagagcgc tgccatcaac cagatcctgg gccgcaggtg gcacgcgctg tcgcgagaag
     1681 agcaggccaa gtactatgag ctggcccgca aggagaggca gctgcacatg cagctatacc
     1741 caggctggtc agcgcgggac aactacggga agaagaagag gcggtcgagg gaaaagcacc
     1801 aagaatccac cacaggagga aaaagaaatg cattcggtac ttacccggag aaggccgctg
     1861 ccccagcccc gttccttccg atgacagtgc tcttgggtac cggagccact aacttctccc
     1921 tgttgaaaca agcaggggat gtcgaagaga atcccgggcc acctaggatg gcaggtggaa
     1981 aagcaggtaa agacagtgga aaagccaaga cgaaagccgt ctccagatct cagagagctg
     2041 gtctgcagtt ccctgtaggt cgtatccaca ggcacctgaa gacacgaacc accagccatg
     2101 gccgtgtcgg agctacagct gcggtttaca gtgctgctat cctggagtat ctcaccgctg
     2161 aggtgctgga gttggctgga aacgcttcta aggatctgaa ggtgaagcga atcactccca
     2221 gacatttaca gctggccatc cgcggagacg aggagctgga ctcgcttatc aaggccacca
     2281 ttgctggagg aggagtcatt cctcacatcc acaagtctct gattggtaag aagggccagc
     2341 agaaaaccgc aggggggatg gtgagcaagg gcgaggagga caacatggcc atcatcaagg
     2401 agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
     2461 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     2521 agggcggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca
     2581 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg
     2641 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
     2701 actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc
     2761 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga
     2821 tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg
     2881 gcggccacta cgacgccgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc
     2941 ccggcgccta caacgtcaac atcaagctgg acatcacctc ccacaacgag gactacacca
     3001 tcgtggaaca gtacgagcgc gccgagggcc gccactccac cggcggcatg gacgagctgt
     3061 acaagtaatg atagctcgag agatctccca gctttcttgt acaaagttgg cattataaga
     3121 aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt
     3181 atttgccatc cagctgatat cccctatagt gagtcgtatt acatggtcat agctgtttcc
     3241 tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata
     3301 tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     3361 catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat
     3421 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
     3481 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     3541 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     3601 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa
     3661 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     3721 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     3781 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     3841 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     3901 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     3961 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     4021 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     4081 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     4141 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc
     4201 attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac
     4261 gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4321 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4381 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4441 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4501 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4561 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4621 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4681 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4741 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4801 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4861 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4921 ttacggttcc tggccttttg ctggcctttt gctcacatgt t
//
