LOCUS       sequence-462701-        4778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A middle entry gateway clone containing V5 tagged human MAFB 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 4778)
  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 4778)
  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 1052 (pME_V5_MAFB_P2A_H2A_mCherry)
FEATURES             Location/Qualifiers
     source          1..4778
                     /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             1131..1154
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1224..1241
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1722..1778
                     /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             2175..2885
                     /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(2320..2338)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2588..2607
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2782..2805
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(2903..3002)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3019..3038)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3020..3038)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3043..3059)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3043..3059)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3161..3180)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3172..3981
                     /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(3239..3258)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4128..4716
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4617..4636
                     /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 atggccgcgg agctgagcat ggggccagag ctgcccacca
      781 gcccgctggc catggagtat gtcaacgact tcgacctgct caagttcgac gtgaagaagg
      841 agccactggg gcgcgcggag cgtccgggca ggccctgcac acgcctgcag ccagccggct
      901 cggtgtcctc cacaccgctc agcactccgt gtagctccgt gccctcgtcg cccagcttca
      961 gcccgaccga acagaagaca cacctcgagg atctgtactg gatggcgagc aactaccagc
     1021 agatgaaccc cgaggcgctc aacctgacgc ccgaggacgc ggtggaagcg ctcatcggct
     1081 cgcacccagt gccacagccg ctgcaaagct tcgacagctt tcgcggcgct caccaccacc
     1141 accatcacca ccaccctcac ccgcaccacg cgtacccggg cgccggcgtg gcccacgacg
     1201 agctgggccc gcacgctcac ccgcaccatc accatcatca ccaagcgtcg ccgccgccgt
     1261 ccagcgccgc tagcccggcg caacagctgc ccactagcca ccccgggccc gggccgcacg
     1321 cgacggcctc ggcgacggcg gcgggcggca acggcagcgt ggaggaccgc ttctccgatg
     1381 accagctcgt gtccatgtcc gtgcgcgagc tgaaccgcca cctgcggggc ttcaccaagg
     1441 acgaggtgat ccgcctgaag cagaagcggc ggaccctgaa gaaccggggc tacgcccagt
     1501 cttgcaggta taaacgcgtc cagcagaagc accacctgga gaatgagaag acgcagctca
     1561 ttcagcaggt ggagcagctt aagcaggagg tgtcccggct ggcccgcgag agagacgcct
     1621 acaaggtcaa gtgcgagaaa ctcgccaact ccggcttcag ggaggcgggc tccaccagcg
     1681 acagcccctc ctctcccgag ttctttctgt gcggtaccgg agccactaac ttctccctgt
     1741 tgaaacaagc aggggatgtc gaagagaatc ccgggccacc taggatggca ggtggaaaag
     1801 caggtaaaga cagtggaaaa gccaagacga aagccgtctc cagatctcag agagctggtc
     1861 tgcagttccc tgtaggtcgt atccacaggc acctgaagac acgaaccacc agccatggcc
     1921 gtgtcggagc tacagctgcg gtttacagtg ctgctatcct ggagtatctc accgctgagg
     1981 tgctggagtt ggctggaaac gcttctaagg atctgaaggt gaagcgaatc actcccagac
     2041 atttacagct ggccatccgc ggagacgagg agctggactc gcttatcaag gccaccattg
     2101 ctggaggagg agtcattcct cacatccaca agtctctgat tggtaagaag ggccagcaga
     2161 aaaccgcagg ggggatggtg agcaagggcg aggaggacaa catggccatc atcaaggagt
     2221 tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg
     2281 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     2341 gcggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg
     2401 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct
     2461 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     2521 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct
     2581 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt
     2641 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg
     2701 gccactacga cgccgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg
     2761 gcgcctacaa cgtcaacatc aagctggaca tcacctccca caacgaggac tacaccatcg
     2821 tggaacagta cgagcgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     2881 agtaatgata gctcgagaga tctcccagct ttcttgtaca aagttggcat tataagaaag
     2941 cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt
     3001 tgccatccag ctgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg
     3061 cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca
     3121 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     3181 attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg
     3241 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg
     3301 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     3361 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     3421 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca
     3481 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     3541 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     3601 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     3661 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt
     3721 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     3781 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     3841 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     3901 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     3961 atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt
     4021 acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttacgcg
     4081 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4141 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4201 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4261 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4321 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4381 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4441 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4501 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4561 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4621 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4681 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4741 cggttcctgg ccttttgctg gccttttgct cacatgtt
//
