LOCUS       sequence-446918-        3925 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible middle entry clone containing a loxP flanked, 
            self inactivating Cre that contains an intron to prevent 
            recombination in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3925)
  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 3925)
  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 iCre-I (JDW 1203)
FEATURES             Location/Qualifiers
     source          1..3925
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    184..283
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(326..359)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      379..388
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             391..411
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          646..778
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     CDS             1577..1603
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(1655..1688)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1718..1817)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1835..1851)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1835..1851)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1848..1870)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1859..1875
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1883..1889,1890..1907,1908..1913))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1928..1949
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2066..2083)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2237..2825)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2317..2336)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3003..3812)
                     /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     3726..3745
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3804..3823
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 acccaaataa tgattttatt ttgactgata gtgacctgtt cgttgcaaca cattgatgag
      241 caatgctttt ttataatgcc aactttgtac aaaaaagcag gctttaatta accgtcttaa
      301 gcctgtaaca accggtacag ttcgaataac ttcgtatagc atacattata cgaagttatg
      361 gtaccgaatt caagcttcgc caccatggtg cccaagaaga agaggaaagt ctccaacctg
      421 ctgactgtgc accaaaacct gcctgccctc cctgtggatg ccacctctga tgaagtcagg
      481 aagaacctga tggacatgtt cagggacagg caggccttct ctgaacacac ctggaagatg
      541 ctcctgtctg tgtgcagatc ctgggctgcc tggtgcaagc tgaacaacag gaaatggttc
      601 cctgctgaac ctgaggatgt gagggactac ctcctgtacc tgcaggtaag tatcaaggtt
      661 acaagacagg tttaaggaga ccaatagaaa ctgggcttgt cgagacagag aagactcttg
      721 cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct ctccacaggc
      781 cagaggcctg gctgtgaaga ccatccaaca gcacctgggc cagctcaaca tgctgcacag
      841 gagatctggc ctgcctcgcc cttctgactc caatgctgtg tccctggtga tgaggagaat
      901 cagaaaggag aatgtggatg ctggggagag agccaagcag gccctggcct ttgaacgcac
      961 tgactttgac caagtcagat ccctgatgga gaactctgac agatgccagg acatcaggaa
     1021 cctggccttc ctgggcattg cctacaacac cctgctgcgc attgccgaaa ttgccagaat
     1081 cagagtgaag gacatctccc gcaccgatgg tgggagaatg ctgatccaca ttggcaggac
     1141 caagaccctg gtgtccacag ctggtgtgga gaaggccctg tccctggggg ttaccaagct
     1201 ggtggagaga tggatctctg tgtctggtgt ggctgatgac cccaacaact acctgttctg
     1261 ccgggtcaga aagaatggtg tggctgcccc ttctgccacc tcccaactgt ccacccgggc
     1321 cctggaaggg atctttgagg ccacccaccg cctgatctat ggtgccaagg atgactctgg
     1381 gcagagatac ctggcctggt ctggccactc tgccagagtg ggtgctgcca gggacatggc
     1441 cagggctggt gtgtccatcc ctgaaatcat gcaggctggt ggctggacca atgtgaacat
     1501 tgtgatgaac tacatcagaa acctggactc tgagactggg gctatggtga ggctgctgga
     1561 ggatggggac ggatcctacc catacgatgt tccagattac gcttaatgag cggccgcact
     1621 tgcaggctgc ctatatcgat acaagcttct ctagataact tcgtatagca tacattatac
     1681 gaagttatac tcgaggcgat cgccgatatc aacgcgtacc cagctttctt gtacaaagtt
     1741 ggcattataa gaaagcattg cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa
     1801 aataaaatca ttatttgagc ttggtgtaat catggtcata gctgtttcct gtgtgaaatt
     1861 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     1921 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     1981 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2041 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2101 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2161 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2221 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2281 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2341 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2401 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2461 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2521 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2581 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2641 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2701 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2761 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2821 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2881 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2941 aaaaatgaag ttttaaatca agcccaatct gaataatgtt acaaccaatt aaccaattct
     3001 gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     3061 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     3121 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     3181 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     3241 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     3301 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     3361 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     3421 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     3481 tattcttcta atacctggaa tgctgttttt ccggggatcg cagtggtgag taaccatgca
     3541 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     3601 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     3661 aacaactctg gcgcatcggg cttcccatac aagcgataga ttgtcgcacc tgattgcccg
     3721 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     3781 ggcctcgacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     3841 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga
     3901 ttttgagaca cgggccagag ctgca
//
