LOCUS       sequence-406396-        3708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ENTR Vector 1 for MultiSite Gateway assembly. Encodes mAG-hGeminin 
            under the control of CMV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3708)
  AUTHORS   Aulicino F, Pelosse M, Toelzer C, Capin J, Ilegems E, Meysami P, 
            Rollarson R, Berggren PO, Dillingham MS, Schaffitzel C, Saleem MA, 
            Welsh GI, Berger I
  TITLE     Highly efficient CRISPR-mediated large DNA docking and multiplexed 
            prime editing using a single baculovirus.
  JOURNAL   Nucleic Acids Res. 2022 Jul 22;50(13):7783-7799. doi: 
            10.1093/nar/gkac587.
  PUBMED    35801912
REFERENCE   2  (bases 1 to 3708)
  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"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2022-07-22- 22"; volume: "50"; issue: 
            "13"; pages: "7783-7799"
COMMENT     Sequence Label: pMMK ENTR 1 CMV mAG h-Geminin
FEATURES             Location/Qualifiers
     source          1..3708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    605..704
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     enhancer        737..1040
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1041..1244
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1195..1215
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1316..1325
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1322..1999
                     /codon_start=1
                     /product="humanized monomeric Azami-Green fluorescent 
                     protein"
                     /label=hmAzami-Green
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLL
                     LEGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             1322..1999
                     /codon_start=1
                     /product="monomeric Azami-Green fluorescent protein 
                     (Karasawa et al., 2003)"
                     /label=mAzami-Green
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLL
                     LEGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             2030..2359
                     /codon_start=1
                     /product="degron consisting of residues 1-110 of human 
                     Geminin (Zielke and Edgar, 2015)"
                     /label=Gem1 (1-110)
                     /translation="MNPSMKQKQEEIKENIKNSSVPRRTLKMIQPSASGSLVGRENELS
                     AGLSKRKHRNDHLTSTTSSPGVIVPESSENKNLGGVTQESFDLMIKENPSSQYWKEVAE
                     KRRKAL"
     polyA_signal    2383..2504
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2420..2439)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2474..2493
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2523..2646
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2742..2761)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2753..3562
                     /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(2820..2839)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcgcaaata atgattttat tttgactgat agtgacctgt tcgttgcaac aaattgataa
      661 gcaatgcttt tttataatgc caactttgta caaaaaagca ggctgtttaa acgatatccc
      721 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      781 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      841 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      901 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      961 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     1021 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     1081 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     1141 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     1201 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggaatt ctgcagtcga
     1261 tcgacggtac cgcgggcccg ggccactagt aacggccgcc agtgtgctgg aattcgccac
     1321 catggtgagc gtgatcaagc ccgagatgaa gatcaagctg tgcatgaggg gcaccgtgaa
     1381 cggccacaac ttcgtgatcg agggcgaggg caagggcaac ccctacgagg gcacccagat
     1441 cctggacctg aacgtgaccg agggcgcccc cctgcccttc gcctacgaca tcctgaccac
     1501 cgtgttccag tacggcaaca gggccttcac caagtacccc gccgacatcc aggactactt
     1561 caagcagacc ttccccgagg gctaccactg ggagaggagc atgacctacg aggaccaggg
     1621 catctgcacc gccaccagca acatcagcat gaggggcgac tgcttcttct acgacatcag
     1681 gttcgacggc accaacttcc cccccaacgg ccccgtgatg cagaagaaga ccctgaagtg
     1741 ggagcccagc accgagaaga tgtacgtgga ggacggcgtg ctgaagggcg acgtgaacat
     1801 gaggctgctg ctggagggcg gcggccacta caggtgcgac ttcaagacca cctacaaggc
     1861 caagaaggag gtgaggctgc ccgacgccca caagatcgac cacaggatcg agatcctgaa
     1921 gcacgacaag gactacaaca aggtgaagct gtacgagaac gccgtggcca ggtactccat
     1981 gctgcccagc caggccaagg gatatccatc acactggcgg ccgctcgaga tgaatcccag
     2041 tatgaagcag aaacaagaag aaatcaaaga gaatataaag aatagttctg tcccaagaag
     2101 aactctgaag atgattcagc cttctgcatc tggatctctt gttggaagag aaaatgagct
     2161 gtccgcaggc ttgtccaaaa ggaaacatcg gaatgaccac ttaacatcta caacttccag
     2221 ccctggggtt attgtcccag aatctagtga aaataaaaat cttggaggag tcacccagga
     2281 gtcatttgat cttatgatta aagaaaatcc atcctctcag tattggaagg aagtggcaga
     2341 aaaacggaga aaggcgctgt aatgaatgca attgttgttg ttaacttgtt tattgcagct
     2401 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     2461 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttagatatc cctaggcccg
     2521 ggcaactttg tataataaag ttgaacgaga aacgtaaaat gatataaata tcaatatatt
     2581 aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc
     2641 actatgctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     2701 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     2761 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     2821 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     2881 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     2941 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     3001 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     3061 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     3121 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     3181 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     3241 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     3301 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     3361 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     3421 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     3481 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     3541 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat
     3601 tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc
     3661 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
