LOCUS       sequence-462696-        4814 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A middle entry gateway clone containing V5 tagged human GATA1 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 4814)
  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 4814)
  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 1050 (pME_V5_GATA1_P2A_H2A_mCherry)
FEATURES             Location/Qualifiers
     source          1..4814
                     /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             1758..1814
                     /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             2211..2921
                     /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(2356..2374)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2624..2643
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2818..2841
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(2939..3038)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3055..3074)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3056..3074)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3079..3095)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3079..3095)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3197..3216)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3208..4017
                     /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(3275..3294)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4164..4752
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4653..4672
                     /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 atcagccacc atgggtaagc ctatccctaa ccctctcctc ggtctcgatt
      721 ctacgggtgg tgctggtggt atggagttcc ctggcctggg gtccctgggg acctcagagc
      781 ccctccccca gtttgtggat cctgctctgg tgtcctccac accagaatca ggggttttct
      841 tcccctctgg gcctgagggc ttggatgcag cagcttcctc cactgccccg agcacagcca
      901 ccgctgcagc tgcggcactg gcctactaca gggacgctga ggcctacaga cactccccag
      961 tctttcaggt gtacccattg ctcaactgta tggaggggat cccagggggc tcaccatatg
     1021 ccggctgggc ctacggcaag acggggctct accctgcctc aactgtgtgt cccacccgcg
     1081 aggactctcc tccccaggcc gtggaagatc tggatggaaa aggcagcacc agcttcctgg
     1141 agactttgaa gacagagcgg ctgagcccag acctcctgac cctgggacct gcactgcctt
     1201 catcactccc tgtccccaat agtgcttatg ggggccctga cttttccagt accttctttt
     1261 ctcccaccgg gagccccctc aattcagcag cctattcctc tcccaagctt cgtggaactc
     1321 tccccctgcc tccctgtgag gccagggagt gtgtgaactg cggagcaaca gccactccac
     1381 tgtggcggag ggacaggaca ggccactacc tatgcaacgc ctgcggcctc tatcacaaga
     1441 tgaatgggca gaacaggccc ctcatccggc ccaagaagcg cctgattgtc agtaaacggg
     1501 caggtactca gtgcaccaac tgccagacga ccaccacgac actgtggcgg agaaatgcca
     1561 gtggggatcc cgtgtgcaat gcctgcggcc tctactacaa gctacaccac cagcactact
     1621 gtggtggctc cgctcagctc atgagggcac agagcatggc ctccagagga ggggtggtgt
     1681 ccttctcctc ttgtagccag aattctggac aacccaagtc tctgggcccc aggcaccccc
     1741 tggcttgcgg taccggagcc actaacttct ccctgttgaa acaagcaggg gatgtcgaag
     1801 agaatcccgg gccacctagg atggcaggtg gaaaagcagg taaagacagt ggaaaagcca
     1861 agacgaaagc cgtctccaga tctcagagag ctggtctgca gttccctgta ggtcgtatcc
     1921 acaggcacct gaagacacga accaccagcc atggccgtgt cggagctaca gctgcggttt
     1981 acagtgctgc tatcctggag tatctcaccg ctgaggtgct ggagttggct ggaaacgctt
     2041 ctaaggatct gaaggtgaag cgaatcactc ccagacattt acagctggcc atccgcggag
     2101 acgaggagct ggactcgctt atcaaggcca ccattgctgg aggaggagtc attcctcaca
     2161 tccacaagtc tctgattggt aagaagggcc agcagaaaac cgcagggggg atggtgagca
     2221 agggcgagga ggacaacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
     2281 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     2341 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg
     2401 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     2461 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     2521 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     2581 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     2641 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     2701 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgcc gaggtcaaga
     2761 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagc
     2821 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgcgccgagg
     2881 gccgccactc caccggcggc atggacgagc tgtacaagta atgatagctc gagagatctc
     2941 ccagctttct tgtacaaagt tggcattata agaaagcatt gcttatcaat ttgttgcaac
     3001 gaacaggtca ctatcagtca aaataaaatc attatttgcc atccagctga tatcccctat
     3061 agtgagtcgt attacatggt catagctgtt tcctggcagc tctggcccgt gtctcaaaat
     3121 ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct
     3181 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcgaggccg
     3241 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
     3301 gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt
     3361 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     3421 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     3481 gcatggttac tcaccactgc gatccccgga aaaacagcat tccaggtatt agaagaatat
     3541 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     3601 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa
     3661 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     3721 cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc
     3781 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     3841 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     3901 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     3961 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca
     4021 gaattggtta attggttgta acactggcag agcattacgc tgacttgacg ggacggcgca
     4081 agctcatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc gtcagacccc
     4141 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4201 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4261 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4321 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4381 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4441 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4501 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4561 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4621 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4681 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4741 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4801 tttgctcaca tgtt
//
