LOCUS       sequence-459820-        5114 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A middle entry gateway clone containing myc-tagged murine dominant 
            negative 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 5114)
  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 5114)
  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 947 (pME-mmTcf4_DN_P2A_mCherry)
FEATURES             Location/Qualifiers
     source          1..5114
                     /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"
     CDS             2058..2114
                     /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             2511..3221
                     /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(2656..2674)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2924..2943
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3118..3141
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(3239..3338)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3355..3374)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3356..3374)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3379..3395)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3379..3395)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3497..3516)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3508..4317
                     /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(3575..3594)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4464..5052
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4953..4972
                     /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 atcccccacc atggcatcaa tgcagaagct gatctcagag gaggacctgc
      721 ttatggccat ggaggcccga attcggtcga ccgagatctc tatgcaagac agctcctccg
      781 attccgaggc ggaaagacgg cctccgcctc gctccgaaag tttccgagat aaatcccggg
      841 aaagtttgga agaagcggcc aagaggcaag atggagggct ctttaagggg ccaccgtatc
      901 ccggctaccc cttcatcatg atccccgacc tgacgagccc ctacctcccc aacggatcgc
      961 tctcgcccac cgcccgaacc ctccattttc aatccggcag cactcattac tctgcgtaca
     1021 aaacgattga acaccagatt gcaattcagt atcttcagat gaaatggcca ctgcttgatg
     1081 tccaagcagg aagcctccag agcagacaaa ccctcaagga tgctcgttcg ccgtcgccag
     1141 cacacatcgt ttcgaacaaa gtaccggtgg tgcaacaccc ccaccatgtc cacccactca
     1201 cgcctctcat cacgtacagc aatgaacact tcaccccggg aaatccacct ccgcacttac
     1261 cagctgacgt agaccccaaa acaggaatcc caaggcctcc gcaccctcca gatatctctc
     1321 catattaccc gctgtcgccc ggcaccgtag gacaaatccc ccatccgcta ggatggttag
     1381 taccacagca aggtcagcct gtgtacccaa tcacgacagg aggattcaga cacccctacc
     1441 ccacagcgct gacagtcaac gcatctatgt ctaggttccc tccccatatg gtccctcccc
     1501 atcacactct gcacacgacc ggcatccctc acccggccat cgtcacaccg acagtcaagc
     1561 aggaatcctc ccagagtgac gtcggctcac tccacagctc aaagcatcag gactccaaaa
     1621 aggaagaaga gaagaagaag ccccacataa agaagcccct taatgcattc atgttgtata
     1681 tgaaagagat gagagcgaag gtggtggccg aatgcacatt gaaagagagt gcagccatca
     1741 accagattct cgggcgcagg tggcacgccc tgtccaggga agaacaggca aaatactacg
     1801 agctggcccg gaaggaacga cagcttcaca tgcagctgta ccctggctgg tctgcacggg
     1861 ataactatgg gaagaagaag aagagaaaaa gagacaagca gccgggggaa accaacgaac
     1921 acagcgaatg tttcctaaat ccttgccttt cgcttcctcc gatcacagac ctgagcgctc
     1981 ctaagaaatg ccgagcgcgc tttggccttg atcaacagaa taactggtgc ggcccctgca
     2041 gtcttcgagg taccggagcc actaacttct ccctgttgaa acaagcaggg gatgtcgaag
     2101 agaatcccgg gccacctagg atggcaggtg gaaaagcagg taaagacagt ggaaaagcca
     2161 agacgaaagc cgtctccaga tctcagagag ctggtctgca gttccctgta ggtcgtatcc
     2221 acaggcacct gaagacacga accaccagcc atggccgtgt cggagctaca gctgcggttt
     2281 acagtgctgc tatcctggag tatctcaccg ctgaggtgct ggagttggct ggaaacgctt
     2341 ctaaggatct gaaggtgaag cgaatcactc ccagacattt acagctggcc atccgcggag
     2401 acgaggagct ggactcgctt atcaaggcca ccattgctgg aggaggagtc attcctcaca
     2461 tccacaagtc tctgattggt aagaagggcc agcagaaaac cgcagggggg atggtgagca
     2521 agggcgagga ggacaacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
     2581 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     2641 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg
     2701 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     2761 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     2821 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     2881 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     2941 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     3001 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgcc gaggtcaaga
     3061 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagc
     3121 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgcgccgagg
     3181 gccgccactc caccggcggc atggacgagc tgtacaagta atgatagctc gagagatctc
     3241 ccagctttct tgtacaaagt tggcattata agaaagcatt gcttatcaat ttgttgcaac
     3301 gaacaggtca ctatcagtca aaataaaatc attatttgcc atccagctga tatcccctat
     3361 agtgagtcgt attacatggt catagctgtt tcctggcagc tctggcccgt gtctcaaaat
     3421 ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct
     3481 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcgaggccg
     3541 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
     3601 gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt
     3661 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     3721 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     3781 gcatggttac tcaccactgc gatccccgga aaaacagcat tccaggtatt agaagaatat
     3841 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     3901 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa
     3961 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     4021 cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc
     4081 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     4141 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     4201 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     4261 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca
     4321 gaattggtta attggttgta acactggcag agcattacgc tgacttgacg ggacggcgca
     4381 agctcatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc gtcagacccc
     4441 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4501 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4561 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4621 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4681 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4741 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4801 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4861 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4921 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4981 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5041 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5101 tttgctcaca tgtt
//
