LOCUS       sequence-446885-        4343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible 3' entry clone containing IRES-FLAG- 
            NLS-mScarlet-I3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4343)
  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 4343)
  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: p3E-IRES-3xNLS-mScarlet-I3-pA (JDW 1360)
FEATURES             Location/Qualifiers
     source          1..4343
                     /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    complement(624..747)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    join(749..1323,1324..1326,1327..1335)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(916..933)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1143..1162
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1339..1404
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1423..1443
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1447..1467
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1471..1491
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1513..2199
                     /codon_start=1
                     /product="bright cysteine-free monomeric red fluorescent 
                     protein with fast and complete maturation (Gadella et al., 
                     2023)"
                     /label=mScarlet3
                     /translation="MDSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYWKQSFPEGFKWERVMIFEDGGT
                     VSVTQDTSLEDGTLIYKVKLRGGNFPPDGPVMQKRTMGWEASTERLYPEDVVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHS
                     TGGSGGS"
     primer_bind     complement(1643..1661)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    2256..2390
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2280..2299)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2334..2353
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(2452..2547)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2584..2603)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2585..2603)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2608..2624)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2608..2624)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2726..2745)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2737..3546
                     /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(2804..2823)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3693..4281
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4182..4201
                     /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 tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtggccc cctctccctc ccccccccct aacgttactg
      781 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat
      841 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc
      901 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag
      961 cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc
     1021 ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac
     1081 ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca
     1141 aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt
     1201 gtatgggatc tgatctgggg cctcggtaca catgctttac atgtgtttag tcgaggttaa
     1261 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat
     1321 aatatggcca caaccatgga ctacaaagac catgacggtg attataaaga tcatgacatc
     1381 gactacaagg atgacgatga caaaggatcc accggtgaat tcccgaagaa gaaaagaaaa
     1441 gtaggaccca agaagaaacg caaagtcgga ccgaagaaga aacgaaaagt cagatctggc
     1501 ggtgctggtg gtatggatag caccgaggca gtgatcaagg agttcatgcg gttcaaggtg
     1561 cacatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     1621 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     1681 tcctgggaca tcctgtcccc tcagttcatg tacggctcca gggccttcat caagcacccc
     1741 gccgacatcc ccgactactg gaagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg
     1801 atgatcttcg aggacggcgg caccgtgtcc gtgacccagg acacctccct ggaggacggc
     1861 accctgatct acaaggtgaa gctccgcggc ggcaacttcc ctcctgacgg ccccgtaatg
     1921 cagaagagga caatgggctg ggaagcatcc accgagcggt tgtaccccga ggacgtcgtg
     1981 ctgaagggcg acattaagat ggccctgcgc ctgaaggacg gcggccgcta cctggcggac
     2041 ttcaagacca cctacaaggc caagaagccc gtgcagatgc ccggcgcctt caacatcgac
     2101 cgcaagttgg acatcacctc ccacaacgag gactacaccg tggtggaaca gtacgaacgc
     2161 tccgtggccc gccactccac cggcggctcc ggtggctccg ctagcggctg atatctataa
     2221 tgaggcgcgc cgtcgacacg cgttaatgat acgtagatcc agacatgata agatacattg
     2281 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     2341 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacatct
     2401 agacattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt ccaactttat
     2461 tatacaaagt tggcattata aaaaagcatt gcttatcaat ttgttgcaac gaacaggtca
     2521 ctatcagtca aaataaaatc attatttgga gctccatggt agcgttaacg cggccgcgat
     2581 atcccctata gtgagtcgta ttacatggtc atagctgttt cctggcagct ctggcccgtg
     2641 tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg aacaataaaa
     2701 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
     2761 tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
     2821 gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca
     2881 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     2941 agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact
     3001 cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt ccaggtatta
     3061 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
     3121 ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct
     3181 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     3241 aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg
     3301 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     3361 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     3421 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     3481 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     3541 ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct gacttgacgg
     3601 gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg
     3661 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3721 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3781 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     3841 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3901 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3961 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4021 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4081 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4141 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4201 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4261 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4321 tgctggcctt ttgctcacat gtt
//
