LOCUS       sequence-462710-        5560 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A middle entry gateway clone containing V5 tagged human TCFL7 / TCF3
            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 5560)
  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 5560)
  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 1035 (pME_V5_TCF7L1_P2A_H2A_mCherry)
FEATURES             Location/Qualifiers
     source          1..5560
                     /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      674..683
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             683..724
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2504..2560
                     /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             2957..3667
                     /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(3102..3120)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3370..3389
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3564..3587
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(3685..3784)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3801..3820)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3802..3820)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3825..3841)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3825..3841)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3943..3962)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3954..4763
                     /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(4021..4040)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4910..5498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5399..5418
                     /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 atcgccacca tgggtaagcc tatccctaac cctctcctcg gtctcgattc
      721 tacgggtggt gctggtggta tgccccagct cggcggcggg ggcggcggcg gcggcggcag
      781 cgggggaggc ggcggctcca gcgccggggc ggccggcgga ggggacgacc tcggggcgaa
      841 cgacgagctg atccccttcc aggacgaggg gggcgaggag caggagccga gcagcgatag
      901 cgcctcggcg cagcgggacc tagacgaggt caagtcgtcc ctggtcaacg agtcggagaa
      961 ccagagcagc agctcggact cggaggcgga gaggcgcccg cagcccgtcc gggacacttt
     1021 ccagaagccg cgggactatt tcgccgaagt gagaaggcct caggacagcg cgttctttaa
     1081 aggacccccg taccctgggt accccttcct gatgatcccg gacctgagca gcccgtacct
     1141 ctccaacgga cccctgtctc ccggaggagc gcgcacctac ctgcagatga aatggcccct
     1201 cctcgatgtc ccctccagcg ccacagtcaa ggacacgagg tcaccatctc cagcacactt
     1261 gtctaataaa gttcctgtcg ttcagcaccc gcatcacatg catccgctga ctcccctcat
     1321 cacctacagc aatgaccact tctcccccgg ctcccctccc acccacctct ccccagagat
     1381 cgatccaaag acaggaatcc cccggccccc tcacccatcc gagctgtcac cgtattaccc
     1441 actctctccc ggagctgtcg gacaaatccc ccaccccctc ggctggctcg tcccacagca
     1501 aggccagccc atgtactccc ttcctcccgg tggcttccgg cacccttacc ccgccctcgc
     1561 catgaacgcc tcgatgtcca gcctggtctc cagtcggttc tctcctcaca tggtggctcc
     1621 tgcccaccct ggcctgccca cctcagggat cccccaccct gccatcgtct cccccatcgt
     1681 caagcaggaa ccggcacccc ccagcctgag ccctgcagtg agcgtgaaat caccagtcac
     1741 cgtgaaaaag gaggaggaaa agaagcccca cgtgaagaag cctctgaatg ccttcatgtt
     1801 gtatatgaag gagatgaggg ccaaggtggt ggctgagtgc accctgaagg aaagtgcagc
     1861 cattaaccag atccttggaa gaaagtggca caacctgtct cgagaagaac aggccaagta
     1921 ctacgagctg gcccggaagg agcggcagct tcactcgcag ctctacccaa cctggtcagc
     1981 ccgggacaac tatggtaaga aaaagaagag gaagagagaa aagcagctgt cccagacaca
     2041 gtcacagcag caagtccagg aggcagaggg tgccctggcc tccaagagca agaagccatg
     2101 tgttcagtac ctgccccccg agaagccctg tgacagccct gcctcctccc acgggagcat
     2161 gctggactcc ccggccactc cctctgcagc tttggcctca ccagctgccc ctgctgccac
     2221 ccattcggag caagcccagc ccctctccct caccaccaaa ccagaaaccc gggcccagct
     2281 ggctctccac tctgccgcct tcctgtcggc taaggctgca gcctcctcct ctaggcagat
     2341 gggcagccag cctcccctcc tgtcccggcc cctccccctt gggtccatgc ccacagctct
     2401 gctggcctct cccccgtcct tccccgccac gctccatgcc caccaggccc tcccggtgct
     2461 acaggcccag cctctttccc tggtcaccaa gtctgcccac ggagccacta acttctccct
     2521 gttgaaacaa gcaggggatg tcgaagagaa tcccgggcca cctaggatgg caggtggaaa
     2581 agcaggtaaa gacagtggaa aagccaagac gaaagccgtc tccagatctc agagagctgg
     2641 tctgcagttc cctgtaggtc gtatccacag gcacctgaag acacgaacca ccagccatgg
     2701 ccgtgtcgga gctacagctg cggtttacag tgctgctatc ctggagtatc tcaccgctga
     2761 ggtgctggag ttggctggaa acgcttctaa ggatctgaag gtgaagcgaa tcactcccag
     2821 acatttacag ctggccatcc gcggagacga ggagctggac tcgcttatca aggccaccat
     2881 tgctggagga ggagtcattc ctcacatcca caagtctctg attggtaaga agggccagca
     2941 gaaaaccgca ggggggatgg tgagcaaggg cgaggaggac aacatggcca tcatcaagga
     3001 gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
     3061 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     3121 gggcggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
     3181 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
     3241 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
     3301 ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
     3361 ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat
     3421 gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg
     3481 cggccactac gacgccgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc
     3541 cggcgcctac aacgtcaaca tcaagctgga catcacctcc cacaacgagg actacaccat
     3601 cgtggaacag tacgagcgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta
     3661 caagtaatga tagctcgaga gatctcccag ctttcttgta caaagttggc attataagaa
     3721 agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta
     3781 tttgccatcc agctgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct
     3841 ggcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat
     3901 catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     3961 atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct gatttatatg
     4021 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg
     4081 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     4141 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     4201 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggaaaaa
     4261 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     4321 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     4381 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     4441 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
     4501 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     4561 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     4621 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     4681 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     4741 tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac tggcagagca
     4801 ttacgctgac ttgacgggac ggcgcaagct catgaccaaa atcccttaac gtgagttacg
     4861 cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4921 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4981 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5041 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5101 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5161 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5221 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5281 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5341 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5401 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5461 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5521 tacggttcct ggccttttgc tggccttttg ctcacatgtt
//
