LOCUS       sequence-447030-        4981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway compatible middle entry clone containing mCerulean3 fused to
            TUBB1A for visualizing tubulin in cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4981)
  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 4981)
  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-mCerulean3-TUBB1A (JDW 1367)
FEATURES             Location/Qualifiers
     source          1..4981
                     /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"
     polyA_signal    695..816
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(706..725)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     760..779
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(2356..3066,3067..3069,3070..3072))
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2398..2419)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2747..2766
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3006..3027
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3069..3078
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3106..3205)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3222..3241)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3223..3241)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3246..3262)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3246..3262)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3364..3383)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3375..4184
                     /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(3442..3461)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4331..4919
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4820..4839
                     /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 aaagcttgcg gccgcactag tcgttaagat acattgatga gtttggacaa
      721 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
      781 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
      841 atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa
      901 tgtggtatgg ctgattatga tcagttatct agatccggtg gatccttagt attcctctcc
      961 ttcttcctca ccctctcctt caacagaatc cacaccaacc tcctcataat ccttctcaag
     1021 ggcagccata tcttcacggg cctctgaaaa ctcgccttcc tccatcccct cacccacgta
     1081 ccagtgaaca aaggcacgct tggcatacat caggtcaaac ttgtggtcca ggcgagccca
     1141 ggcctcagca atggctgtgg tgttgctcag catgcacaca gctctctgta ccttggccag
     1201 gtctccacca ggcaccacag tgggaggctg gtagttgatg ccaaccttga agccagtggg
     1261 gcaccaatcc acaaactgga tgctgcgctt ggttttgatg gtggcaatgg cagcattgac
     1321 atctttggga accacgtcac cacggtacaa caggcagcaa gccatgtatt taccatggcg
     1381 agggtcacat ttcaccatct ggttggctgg ctcaaagcaa gcattggtga tctctgctac
     1441 agaaagctgt tcatggtagg ctttctcagc agagatgaca ggggcatatg tggccagagg
     1501 gaagtggatg cgggggtagg gcaccaggtt ggtctggaat tctgtcaggt caacattcag
     1561 ggctccatca aatctcaggg aagcagtgat ggaggacaca atctggctaa taaggcggtt
     1621 aaggttagtg taggttgggc gctcgatatc gaggtttcta cgacagatgt catagatggc
     1681 ctcattgtct accatgaagg cacaatcaga gtgctccagg gtggtgtggg tggtgaggat
     1741 ggagttgtag ggctcaacta cagctgtgga aacctggggt gctgggtaaa tggagaactc
     1801 cagcttggac ttcttgccat aatcaactga gagacgttcc atgagcaggg aggtgaaccc
     1861 agaaccagtt cccccaccaa agctgtggaa aaccaagaag ccctgaagac cggtgcactg
     1921 gtcagccagc ttgcgaattc ggtccaacac aaggtcaatg atctccttgc caatggtgta
     1981 gtgccctcgg gcatagttat tggcagcatc ttccttgcct gtgatgagct gctcagggtg
     2041 gaagagctgg cggtaggtgc cagtgcgaac ttcatcaatg actgtgggtt ccaagtctac
     2101 aaacacagcc cggggcacgt gcttgccagc gcccgtctca ctgaagaagg tgttgaagga
     2161 gtcatctcct cccccaatgg tcttgtcact tggcatctgg ccatcgggct ggatgccgtg
     2221 ttccaggcag tagagctccc agcaggcatt gccaatctgg acaccagcct ggccaacgtg
     2281 gatggagatg cactcacgca cgcttccgct tccgccggat gcgctgcctc caccgctgcc
     2341 agatctgagt ccggacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac
     2401 gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gcttggactg
     2461 ggtgctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg
     2521 ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgcagttga ggccgaagtt
     2581 ggccttgatg ccgttcttct gcttgtcggc ggtgatatag acgttgccgt ggatggcgtt
     2641 gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc
     2701 gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc
     2761 gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa
     2821 gaagtcgtgc tgcttcatgt ggtcggggta gcgggcgaag cactgcacgc cccagctcag
     2881 ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag
     2941 ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc
     3001 gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc
     3061 cttgctcacc atggtggcgt gcctcgagtt aattaaggta cctgcaccca gctttcttgt
     3121 acaaagttgg cattataaga aagcattgct tatcaatttg ttgcaacgaa caggtcacta
     3181 tcagtcaaaa taaaatcatt atttgccatc cagctgatat cccctatagt gagtcgtatt
     3241 acatggtcat agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc tgatgttaca
     3301 ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta
     3361 atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca
     3421 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     3481 cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     3541 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     3601 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     3661 ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     3721 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     3781 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     3841 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     3901 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     3961 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     4021 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     4081 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     4141 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt
     4201 ggttgtaaca ctggcagagc attacgctga cttgacggga cggcgcaagc tcatgaccaa
     4261 aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca
     4321 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4381 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4441 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     4501 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4561 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4621 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4681 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4741 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4801 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4861 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4921 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4981 t
//
