LOCUS       sequence-462718-        5207 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A middle entry gateway clone containing V5 tagged human ZIC3 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 5207)
  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 5207)
  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 1394 (pME-ZIC3-P2A-H2A-mCherry)
FEATURES             Location/Qualifiers
     source          1..5207
                     /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             999..1028
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1002..1031
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2151..2207
                     /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             2604..3314
                     /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(2749..2767)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3017..3036
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3211..3234
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(3332..3431)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3448..3467)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3449..3467)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3472..3488)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3472..3488)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3590..3609)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3601..4410
                     /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(3668..3687)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4557..5145
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5046..5065
                     /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 atgggaaaac ctataccaaa tcccttgctg ggcctggact
      721 caacaggtgg tgctggtggt atgacgatgc tcctggacgg aggcccgcag ttccctgggc
      781 tgggagtggg cagcttcggc gcgccgcgcc accacgagat gcccaaccgt gagccggcag
      841 gcatggggct gaatcccttc ggggactcaa cccacgccgc cgccgccgcc gccgccgccg
      901 ctgccttcaa gctgagccct gccgcggcgc acgatctatc ttcaggccag agctcggctt
      961 tcacgccgca gggttcgggc tacgccaacg ccctgggcca ccatcaccac caccatcacc
     1021 atcatcacca caccagccag gtgcccagct acggtggcgc tgcctctgcc gccttcaact
     1081 caacgcgcga gtttctgttc cgccagcgca gctccgggct cagtgaggcg gcctcgggtg
     1141 gcgggcagca cgggctcttc gccggctcgg cgagcagcct gcatgctcca gctggcatcc
     1201 ccgagccccc tagctacttg ctgtttcccg ggctgcatga gcagggcgct gggcacccgt
     1261 cgcccacagg gcacgtggac aacaaccagg tccacctggg gctgcgtggg gagctgttcg
     1321 gccgtgctga cccataccgc ccagtggcca gcccgcgcac ggacccctac gcggccggcg
     1381 ctcagtttcc taactacagc cccatgaaca tgaacatggg agtgaacgtg gcggcccacc
     1441 acgggcccgg cgccttcttc cgttatatgc ggcagcctat caagcaggag ctgtcgtgca
     1501 agtggatcga cgaggctcag ctgagccggc ccaagaagag ctgcgaccgg accttcagca
     1561 ccatgcatga gctggtgaca catgtcacca tggagcatgt ggggggcccg gagcagaaca
     1621 accacgtctg ctactgggag gagtgccccc gggagggcaa gtctttcaag gcgaagtaca
     1681 aactggtcaa ccacatccga gtgcacacgg gcgagaagcc cttcccatgc cccttcccgg
     1741 gctgcgggaa gatctttgcc cgttctgaga acctcaagat ccacaagagg acccacacag
     1801 gtgagaaacc tttcaaatgt gaatttgaag gctgtgacag acgctttgcc aacagcagcg
     1861 accgtaagaa gcacatgcat gtgcatacct cggacaagcc ctatatctgc aaagtgtgcg
     1921 acaagtccta cacgcacccg agctccctgc gcaaacacat gaaggttcat gaatctcaag
     1981 ggtcagattc ctcccctgct gccagttcag gctatgaatc ttccactcca cccgctatag
     2041 cttctgcaaa cagtaaagat accactaaaa ccccttctgc agttcaaact agcaccagcc
     2101 acaaccctgg acttcctcct aattttaacg aatggtacgt cggtaccgga gccactaact
     2161 tctccctgtt gaaacaagca ggggatgtcg aagagaatcc cgggccacct aggatggcag
     2221 gtggaaaagc aggtaaagac agtggaaaag ccaagacgaa agccgtctcc agatctcaga
     2281 gagctggtct gcagttccct gtaggtcgta tccacaggca cctgaagaca cgaaccacca
     2341 gccatggccg tgtcggagct acagctgcgg tttacagtgc tgctatcctg gagtatctca
     2401 ccgctgaggt gctggagttg gctggaaacg cttctaagga tctgaaggtg aagcgaatca
     2461 ctcccagaca tttacagctg gccatccgcg gagacgagga gctggactcg cttatcaagg
     2521 ccaccattgc tggaggagga gtcattcctc acatccacaa gtctctgatt ggtaagaagg
     2581 gccagcagaa aaccgcaggg gggatggtga gcaagggcga ggaggacaac atggccatca
     2641 tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg
     2701 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
     2761 tgaccaaggg cggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg
     2821 gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc
     2881 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga
     2941 cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca
     3001 acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg
     3061 agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga
     3121 aggacggcgg ccactacgac gccgaggtca agaccaccta caaggccaag aagcccgtgc
     3181 agctgcccgg cgcctacaac gtcaacatca agctggacat cacctcccac aacgaggact
     3241 acaccatcgt ggaacagtac gagcgcgccg agggccgcca ctccaccggc ggcatggacg
     3301 agctgtacaa gtaatgatag ctcgagagat ctcccagctt tcttgtacaa agttggcatt
     3361 ataagaaagc attgcttatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa
     3421 atcattattt gccatccagc tgatatcccc tatagtgagt cgtattacat ggtcatagct
     3481 gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa
     3541 aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     3601 atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat
     3661 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc
     3721 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     3781 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     3841 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     3901 ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     3961 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     4021 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     4081 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     4141 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     4201 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     4261 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     4321 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
     4381 tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg
     4441 cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc ccttaacgtg
     4501 agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4561 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4621 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4681 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4741 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4801 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4861 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4921 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4981 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5041 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5101 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5161 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtt
//
