LOCUS       sequence-484477-        4267 bp    DNA     circular SYN 25-AUG-2025
DEFINITION  Gateway middle entry clone containing H2B-mStayGold with WPRE at the
            3' end; Nuclear green fluorescent reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4267)
  AUTHORS   Gillespie W, Zhang Y, Ruiz OE, Cerda J, Ortiz-Guzman J, Turner WD, 
            Largoza G, Sherman M, 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 with diverse applications in vertebrate model 
            systems.
  JOURNAL   bioRxiv [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267.
  PUBMED    39026881
REFERENCE   2  (bases 1 to 4267)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Jul 13:2024.07.13.603267. doi: 
            10.1101/2024.07.13.603267."
COMMENT     Sequence Label: pME-H2B-mStayGold-WPRE (JDW 1384)
FEATURES             Location/Qualifiers
     source          1..4267
                     /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             694..1071
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             1078..1755
                     /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="MVSTGEELFTGVVPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMDVSLPNEVQHIPIDDGVECTVTL
                     QYPLLSDESKCVEAYQNTIIKPLHNQPAPDVPFHWIRKQYTQSKDDTEERDHIIQSETL
                     EAHL"
     misc_feature    1771..2359
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1824..1844)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2242..2253)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(2392..2491)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2508..2527)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2509..2527)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2532..2548)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2532..2548)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2650..2669)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2661..3470
                     /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(2728..2747)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3617..4205
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4106..4125
                     /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 agcaggctgg gatccactag tgctagcgcc accatgccag agccagcgaa gtctgctccc
      721 gccccgaaaa agggctccaa gaaggcggtg actaaggcgc agaagaaagg cggcaagaag
      781 cgcaagcgca gccgcaagga gagctattcc atctatgtgt acaaggttct gaagcaggtc
      841 caccctgaca ccggcatttc gtccaaggcc atgggcatca tgaattcgtt tgtgaacgac
      901 attttcgagc gcatcgcagg tgaggcttcc cgcctggcgc attacaacaa gcgctcgacc
      961 atcacctcca gggagatcca gacggccgtg cgcctgctgc tgcctgggga gttggccaag
     1021 cacgccgtgt ccgagggtac taaggccatc accaagtaca ccagcgctaa gagatctatg
     1081 gtgtctacag gcgaggagct gtttaccggc gtggtgccct tcaagttcca gctgaagggc
     1141 accatcaacg gcaagagctt caccgtggaa ggcgagggcg agggcaatag ccacgagggc
     1201 agccacaaag gcaagtacgt gtgcaccagc ggcaaactgc caatgtcttg ggccgccctg
     1261 ggaactagct tcggctatgg catgaagtac tacaccaagt accccagcgg cctgaagaac
     1321 tggttccacg aggtgatgcc cgagggcttc acctacgaca gacacatcca gtacaagggc
     1381 gacggcagca tccacgccaa gcaccagcac ttcatgaaga acggcaccta ccacaacatc
     1441 gtggagttca ccggccagga cttcaaggag aacagccccg tgctgaccgg cgacatggac
     1501 gtgagcctgc ccaacgaggt gcagcacatc cccatagatg acggcgtgga gtgcacagtg
     1561 accctgcagt accctctgct gagcgacgag agcaagtgcg tggaggccta ccagaacacc
     1621 atcatcaagc ccctgcacaa tcagccagcc cccgatgtgc cattccactg gatcagaaag
     1681 cagtacaccc agagcaagga cgacaccgag gagagagacc acatcatcca gagcgagacc
     1741 ctggaggccc acctgtaaaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     1801 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     1861 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     1921 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     1981 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     2041 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     2101 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     2161 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     2221 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     2281 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     2341 ctttgggccg cctccccgca tcgatggcgc gccacgcgtg ttaactctag aacccagctt
     2401 tcttgtacaa agttggcatt ataagaaagc attgcttatc aatttgttgc aacgaacagg
     2461 tcactatcag tcaaaataaa atcattattt gccatccagc tgatatcccc tatagtgagt
     2521 cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat
     2581 gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa
     2641 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa
     2701 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     2761 caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac
     2821 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     2881 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     2941 tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt
     3001 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     3061 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     3121 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     3181 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
     3241 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     3301 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     3361 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     3421 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg
     3481 ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat
     3541 gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa
     3601 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3661 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3721 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     3781 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3841 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3901 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3961 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4021 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4081 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4141 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4201 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4261 acatgtt
//
