LOCUS       sequence-456038-        4401 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible middle entry clone containing EGFP P2A iCre 
            (codon optimized cre).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4401)
  AUTHORS   Gillespie WB 3rd, Zhang Y, Ruiz OE, Cerda J 3rd, Ortiz-Guzman J, 
            Sherman M, Turner WD, Largoza G, Mosser LE, Fujimoto E, Chien CB, 
            Kwan KM, Arenkiel BR, Devine WP, Wythe JD
  TITLE     MultiSite Assembly of Gateway Induced Clones (MAGIC): a flexible 
            cloning toolbox for use in vertebrate model systems.
  JOURNAL   Development. 2025 Nov 15;152(22):dev204308. doi: 10.1242/dev.204308.
            Epub 2025 Nov 13.
  PUBMED    41230856
REFERENCE   2  (bases 1 to 4401)
  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: 
            "Development."; date: "2025-11-15"; volume: "152(22):dev204308. 
            doi"; pages: " 10.1242/dev.204308. Epub 2025 Nov 13"
COMMENT     Sequence Label: pME-EGFP-P2A-iCre (JDW 921)
FEATURES             Location/Qualifiers
     source          1..4401
                     /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      669..678
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(675..677,678..680,681..1391)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(675..677,678..680,681..1391)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(720..741)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(981..1000)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1328..1349
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1407..1463
                     /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             1476..1496
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(2526..2625)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2642..2661)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2643..2661)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2666..2682)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2666..2682)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2784..2803)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2795..3604
                     /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(2862..2881)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3751..4339
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4240..4259
                     /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 agcaggctgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      721 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      781 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      841 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      901 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      961 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1021 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1081 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1141 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1201 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1261 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     1321 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1381 agctgtacaa gctcgaggga agcggagcca ctaacttctc cctgttgaaa caagcagggg
     1441 atgtcgaaga gaatcccggg ccatctagaa tggtgcccaa gaagaagagg aaagtctcca
     1501 acctgctgac tgtgcaccaa aacctgcctg ccctccctgt ggatgccacc tctgatgaag
     1561 tcaggaagaa cctgatggac atgttcaggg acaggcaggc cttctctgaa cacacctgga
     1621 agatgctcct gtctgtgtgc agatcctggg ctgcctggtg caagctgaac aacaggaaat
     1681 ggttccctgc tgaacctgag gatgtgaggg actacctcct gtacctgcaa gccagaggcc
     1741 tggctgtgaa gaccatccaa cagcacctgg gccagctcaa catgctgcac aggagatctg
     1801 gcctgcctcg cccttctgac tccaatgctg tgtccctggt gatgaggaga atcagaaagg
     1861 agaatgtgga tgctggggag agagccaagc aggccctggc ctttgaacgc actgactttg
     1921 accaagtcag atccctgatg gagaactctg acagatgcca ggacatcagg aacctggcct
     1981 tcctgggcat tgcctacaac accctgctgc gcattgccga aattgccaga atcagagtga
     2041 aggacatctc ccgcaccgat ggtgggagaa tgctgatcca cattggcagg accaagaccc
     2101 tggtgtccac agctggtgtg gagaaggccc tgtccctggg ggttaccaag ctggtggaga
     2161 gatggatctc tgtgtctggt gtggctgatg accccaacaa ctacctgttc tgccgggtca
     2221 gaaagaatgg tgtggctgcc ccttctgcca cctcccaact gtccacccgg gccctggaag
     2281 ggatctttga ggccacccac cgcctgatct atggtgccaa ggatgactct gggcagagat
     2341 acctggcctg gtctggccac tctgccagag tgggtgctgc cagggacatg gccagggctg
     2401 gtgtgtccat ccctgaaatc atgcaggctg gtggctggac caatgtgaac attgtgatga
     2461 actacatcag aaacctggac tctgagactg gggccatggt gaggctgctc gaggatgggg
     2521 actaaaccca gctttcttgt acaaagttgg cattataaga aagcattgct tatcaatttg
     2581 ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgatat
     2641 cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc
     2701 tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact
     2761 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     2821 gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     2881 taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga
     2941 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     3001 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     3061 tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga
     3121 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     3181 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
     3241 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     3301 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     3361 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     3421 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     3481 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     3541 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     3601 ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga
     3661 cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc
     3721 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3781 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3841 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     3901 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3961 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4021 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4081 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4141 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4201 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4261 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4321 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4381 ctggcctttt gctcacatgt t
//
