LOCUS       sequence-446932-        4380 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway-compatible middle entry clone containing mScarlet-I 
            P2A-iCre (codon optimized).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4380)
  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 4380)
  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-mScarlet-I-P2A-iCre (JDW 917)
FEATURES             Location/Qualifiers
     source          1..4380
                     /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             675..1370
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(808..826)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1076..1095
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1386..1442
                     /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             1455..1475
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(2505..2604)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2621..2640)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2622..2640)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2645..2661)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2645..2661)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2763..2782)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2774..3583
                     /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(2841..2860)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3730..4318
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4219..4238
                     /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 aggcagtgat caaggagttc atgcggttca
      721 aggtgcacat ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg
      781 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
      841 ccttctcctg ggacatcctg tcccctcagt tcatgtacgg ctccagggcc ttcaccaagc
      901 accccgccga catccccgac tactataagc agtccttccc cgagggcttc aagtgggagc
      961 gcgtgatgaa cttcgaggac ggcggcgccg tgaccgtgac ccaggacacc tccctggagg
     1021 acggcaccct gatctacaag gtgaagctcc gcggcaccaa cttccctcct gacggccccg
     1081 taatgcagaa gaagacaatg ggctgggaag cgtccaccga gcggttgtac cccgaggacg
     1141 gcgtgctgaa gggcgacatt aagatggccc tgcgcctgaa ggacggcggc cgctacctgg
     1201 cggacttcaa gaccacctac aaggccaaga agcccgtgca gatgcccggc gcctacaacg
     1261 tcgaccgcaa gttggacatc acctcccaca acgaggacta caccgtggtg gaacagtacg
     1321 aacgctccga gggccgccac tccaccggcg gcatggacga gctgtacaag ctcgagggaa
     1381 gcggagccac taacttctcc ctgttgaaac aagcagggga tgtcgaagag aatcccgggc
     1441 catctagaat ggtgcccaag aagaagagga aagtctccaa cctgctgact gtgcaccaaa
     1501 acctgcctgc cctccctgtg gatgccacct ctgatgaagt caggaagaac ctgatggaca
     1561 tgttcaggga caggcaggcc ttctctgaac acacctggaa gatgctcctg tctgtgtgca
     1621 gatcctgggc tgcctggtgc aagctgaaca acaggaaatg gttccctgct gaacctgagg
     1681 atgtgaggga ctacctcctg tacctgcaag ccagaggcct ggctgtgaag accatccaac
     1741 agcacctggg ccagctcaac atgctgcaca ggagatctgg cctgcctcgc ccttctgact
     1801 ccaatgctgt gtccctggtg atgaggagaa tcagaaagga gaatgtggat gctggggaga
     1861 gagccaagca ggccctggcc tttgaacgca ctgactttga ccaagtcaga tccctgatgg
     1921 agaactctga cagatgccag gacatcagga acctggcctt cctgggcatt gcctacaaca
     1981 ccctgctgcg cattgccgaa attgccagaa tcagagtgaa ggacatctcc cgcaccgatg
     2041 gtgggagaat gctgatccac attggcagga ccaagaccct ggtgtccaca gctggtgtgg
     2101 agaaggccct gtccctgggg gttaccaagc tggtggagag atggatctct gtgtctggtg
     2161 tggctgatga ccccaacaac tacctgttct gccgggtcag aaagaatggt gtggctgccc
     2221 cttctgccac ctcccaactg tccacccggg ccctggaagg gatctttgag gccacccacc
     2281 gcctgatcta tggtgccaag gatgactctg ggcagagata cctggcctgg tctggccact
     2341 ctgccagagt gggtgctgcc agggacatgg ccagggctgg tgtgtccatc cctgaaatca
     2401 tgcaggctgg tggctggacc aatgtgaaca ttgtgatgaa ctacatcaga aacctggact
     2461 ctgagactgg ggccatggtg aggctgctcg aggatgggga ctaaacccag ctttcttgta
     2521 caaagttggc attataagaa agcattgctt atcaatttgt tgcaacgaac aggtcactat
     2581 cagtcaaaat aaaatcatta tttgccatcc agctgatatc ccctatagtg agtcgtatta
     2641 catggtcata gctgtttcct ggcagctctg gcccgtgtct caaaatctct gatgttacat
     2701 tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa
     2761 tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa
     2821 catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc
     2881 gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa
     2941 aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt
     3001 tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac
     3061 cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga
     3121 aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa
     3181 ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa
     3241 cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt
     3301 ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga
     3361 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg
     3421 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga
     3481 gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat
     3541 gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg
     3601 gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcgcaagct catgaccaaa
     3661 atcccttaac gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3721 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3781 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3841 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     3901 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3961 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4021 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4081 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4141 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4201 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4261 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4321 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
//
