LOCUS       sequence-447100-        3842 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway compatible middle entry clone contianing Histone H2B fusion 
            to V5-tagged (n2)StayGold(C4) (Nuclear StayGold fluorescent 
            reporter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3842)
  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 3842)
  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-H2B-V5-n2StayGoldC4 (JDW 1354)
FEATURES             Location/Qualifiers
     source          1..3842
                     /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"
     CDS             687..1064
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     CDS             1080..1121
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1200..1877
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ando et al., 2023)"
                     /label=mStayGold(J)
                     /note="includes multiple mutations plus an N-terminal 
                     iinker sequence"
                     /translation="MVSKGEELFTGVTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNEVQHIPRDDGVECPVTL
                     LYPLLSDKSKCVEAYQNTIIKPLHNQPAPDVPYHWIRKQYTQSKDDTEERDHIIQSETL
                     EAHL"
     protein_bind    complement(1967..2066)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2083..2102)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2084..2102)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2107..2123)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2107..2123)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2225..2244)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2236..3045
                     /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(2303..2322)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3192..3780
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3681..3700
                     /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 agcaggctac tagtgctagc gccaccatgc ctgaaccctc taagtctgct ccagccccta
      721 aaaagggttc taagaaggct atcactaagg cgcagaagaa ggatggtaag aagcgtaagc
      781 gcagccgcaa ggagagctat tctatctatg tgtacaaggt tctgaagcag gtccaccccg
      841 acaccggcat ctcatccaag gccatgggga tcatgaactc cttcgtcaac gacatcttcg
      901 agcgcatcgc gggcgaggct tctcgcctgg ctcactacaa taagcgctcg accatcacct
      961 ccagggagat tcagacggct gtgcgcctgc tgctgcctgg ggagctggct aagcatgctg
     1021 tgtccgaggg cactaaggca gttaccaagt acactagctc taagggtggt gctggtggtg
     1081 gtaagcctat ccctaaccct ctcctcggtc tcgattctac gggatccacc ggtgaattcg
     1141 atggtggagg cggttcaggc ggaggtggct ctggcggtgg cggatcgcca tcaggtacca
     1201 tggtgagcaa gggcgaggag ctgtttacag gcgtgacccc cttcaagttc cagctgaagg
     1261 gcaccatcaa cggcaagagc ttcaccgtgg aaggcgaggg cgagggcaat agccacgagg
     1321 gcagccacaa aggcaagtac gtgtgcacca gcggcaaact gccaatgtct tgggccgccc
     1381 tgggaactag cttcggctat ggcatgaagt actacaccaa gtaccccagc ggcctgaaga
     1441 actggttcca cgaggtgatg cccgagggct tcacctacga cagacacatc cagtacaagg
     1501 gcgacggcag catccacgcc aagcaccagc acttcatgaa gaacggcacc taccacaaca
     1561 tcgtggagtt caccggccag gacttcaagg agaacagccc cgtgctgacc ggcgacatga
     1621 acgtgagcct gcccaacgag gtgcagcaca tccccagaga tgacggcgtg gagtgcccag
     1681 tgaccctgct gtaccctctg ctgagcgaca agagcaagtg cgtggaggcc taccagaaca
     1741 ccatcatcaa gcccctgcac aatcagccag cccccgatgt gccataccac tggatcagaa
     1801 agcagtacac ccagagcaag gacgacaccg aggagagaga ccacatcatc cagagcgaga
     1861 ccctggaggc ccacctgcca tggcacgagc cttctgcttc tgccgtgtga tatctataat
     1921 gaggcgcgcc gtcgacgtta acacgcgtta atgatacgta tctagaaccc agctttcttg
     1981 tacaaagttg gcattataag aaagcattgc ttatcaattt gttgcaacga acaggtcact
     2041 atcagtcaaa ataaaatcat tatttgccat ccagctgata tcccctatag tgagtcgtat
     2101 tacatggtca tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac
     2161 attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt
     2221 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg attaaattcc
     2281 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     2341 gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     2401 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     2461 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     2521 accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc tgattcaggt
     2581 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     2641 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     2701 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     2761 gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt
     2821 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     2881 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     2941 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     3001 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat
     3061 tggttgtaac actggcagag cattacgctg acttgacggg acggcgcaag ctcatgacca
     3121 aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc
     3181 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3241 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3301 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     3361 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3421 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3481 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3541 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3601 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3661 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3721 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3781 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3841 tt
//
