LOCUS       sequence-462695-        5174 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A middle entry gateway clone containing V5 tagged human TCF7L2 / 
            TCF4 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 5174)
  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 5174)
  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 1036 (pME_V5_TCF7L2_P2A_H2A_mCherry)
FEATURES             Location/Qualifiers
     source          1..5174
                     /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             2118..2174
                     /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             2571..3281
                     /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(2716..2734)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2984..3003
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3178..3201
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(3299..3398)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3415..3434)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3416..3434)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3439..3455)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3439..3455)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3557..3576)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3568..4377
                     /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(3635..3654)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4524..5112
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5013..5032
                     /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 atgccgcagc tgaacggcgg tggaggggat gacctaggcg
      781 ccaacgacga actgatttcc ttcaaagacg agggcgaaca ggaggagaag agctccgaaa
      841 actcctcggc agagagggat ttagctgatg tcaaatcgtc tctagtcaat gaatcagaaa
      901 cgaatcaaaa cagctcctcc gattccgagg cggaaagacg gcctccgcct cgctccgaaa
      961 gtttccgaga caaatcccgg gaaagtttgg aagaagcggc caagaggcaa gatggagggc
     1021 tctttaaggg gccaccgtat cccggctacc ccttcatcat gatccccgac ctgacgagcc
     1081 cctacctccc caacggatcg ctctcgccca ccgcccgaac ctatctccag atgaaatggc
     1141 cactgcttga tgtccaggca gggagcctcc agagtagaca agccctcaag gatgcccggt
     1201 ccccatcacc ggcacacatt gtctctaaca aagtgccagt ggtgcagcac cctcaccatg
     1261 tccaccccct cacgcctctt atcacgtaca gcaatgaaca cttcacgccg ggaaacccac
     1321 ctccacactt accagccgac gtagacccca aaacaggaat cccacggcct ccgcaccctc
     1381 cagatatatc cccgtattac ccactatcgc ctggcaccgt aggacaaatc ccccatccgc
     1441 taggatggca aggtcaacca gtgtacccaa tcacgacagg aggattcaga cacccctacc
     1501 ccacagctct gaccgtcaat gcttccatgt ccaggttccc tccccatatg gtcccaccac
     1561 atcatacgct acacacgacg ggcattccgc atccggccat agtcacacca acagtcaaac
     1621 aggaatcgtc ccagagtgat gtcggctcac tccatagttc aaagcatcag gactccaaaa
     1681 aggaagaaga aaagaagaag ccccacataa agaaacctct taatgcattc atgttgtata
     1741 tgaaggaaat gagagcaaag gtcgtagctg agtgcacgtt gaaagaaagc gcggccatca
     1801 accagatcct tgggcggagg tggcatgcac tgtccagaga agagcaagcg aaatactacg
     1861 agctggcccg gaaggagcga cagcttcata tgcaactgta ccccggctgg tccgcgcggg
     1921 ataactatgg aaagaagaag aagaggaaaa gggacaagca gccgggagag accaatgaac
     1981 acagcgaatg tttcctaaat ccttgccttt cacttcctcc gattacagga gaaaaaaaaa
     2041 gtgcgttcgc tacatacaag gtgaaggcag ctgcctcagc ccaccctctt cagatggaag
     2101 cttactgcgg taccggagcc actaacttct ccctgttgaa acaagcaggg gatgtcgaag
     2161 agaatcccgg gccacctagg atggcaggtg gaaaagcagg taaagacagt ggaaaagcca
     2221 agacgaaagc cgtctccaga tctcagagag ctggtctgca gttccctgta ggtcgtatcc
     2281 acaggcacct gaagacacga accaccagcc atggccgtgt cggagctaca gctgcggttt
     2341 acagtgctgc tatcctggag tatctcaccg ctgaggtgct ggagttggct ggaaacgctt
     2401 ctaaggatct gaaggtgaag cgaatcactc ccagacattt acagctggcc atccgcggag
     2461 acgaggagct ggactcgctt atcaaggcca ccattgctgg aggaggagtc attcctcaca
     2521 tccacaagtc tctgattggt aagaagggcc agcagaaaac cgcagggggg atggtgagca
     2581 agggcgagga ggacaacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
     2641 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     2701 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg
     2761 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     2821 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     2881 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     2941 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     3001 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     3061 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgcc gaggtcaaga
     3121 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagc
     3181 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgcgccgagg
     3241 gccgccactc caccggcggc atggacgagc tgtacaagta atgatagctc gagagatctc
     3301 ccagctttct tgtacaaagt tggcattata agaaagcatt gcttatcaat ttgttgcaac
     3361 gaacaggtca ctatcagtca aaataaaatc attatttgcc atccagctga tatcccctat
     3421 agtgagtcgt attacatggt catagctgtt tcctggcagc tctggcccgt gtctcaaaat
     3481 ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct
     3541 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcgaggccg
     3601 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
     3661 gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt
     3721 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     3781 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     3841 gcatggttac tcaccactgc gatccccgga aaaacagcat tccaggtatt agaagaatat
     3901 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     3961 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa
     4021 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     4081 cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc
     4141 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     4201 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     4261 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     4321 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca
     4381 gaattggtta attggttgta acactggcag agcattacgc tgacttgacg ggacggcgca
     4441 agctcatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc gtcagacccc
     4501 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4561 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4621 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4681 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4741 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4801 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4861 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4921 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4981 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5041 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5101 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5161 tttgctcaca tgtt
//
