LOCUS       sequence-447342-        3431 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible middle entry clone containing a loxP flanked 
            SV40 polyA, bGH polyA, and SV40 pA stop cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3431)
  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 3431)
  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-lox-3xSTOP-lox (JDW 869)
FEATURES             Location/Qualifiers
     source          1..3431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    33..132
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(133..166)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        186..204
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     186..203
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     polyA_signal    205..339
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(459..475)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(459..475)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     polyA_signal    476..587
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    679..813
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(899..919)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(899..917)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(938..954)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(951..973)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    962..978
                     /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)."
     protein_bind    complement(986..1019)
                     /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(1020..1119)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1137..1155)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1160..1176)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1278..1297)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1289..2098
                     /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(1356..1375)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2245..2833
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2734..2753
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2987..3004
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3163..3190)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3282..3368)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(3417..3431,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(3431,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cccaaataat gattttattt tgactgatag
       61 tgacctgttc gttgcaacaa attgatgagc aatgcttttt tataatgcca actttgtaca
      121 aaaaagcagg ctataacttc gtatagcata cattatacga agttatcgga gcaagcttgg
      181 atcccattta ggtgacacta tagaaacttg tttattgcag cttataatgg ttacaaataa
      241 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
      301 ttgtccaaac tcatcaatgt atcttatcat gtctggatct gcgacctcga gggatcataa
      361 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
      421 tgaacctgaa acataaaatg aatgcaattg ttgttgttga tccactagtt ctagactgtg
      481 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
      541 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcatc gagggggggc
      601 ccggtaacca ccgcggtggc ggcgatctcg cggccgcaag taaagcggcc ctagaactat
      661 agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc
      721 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
      781 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
      841 tttcaggttc agggggaggt gtgggaggtt ttttgggccc ggtacccagc ttttgttccc
      901 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
      961 attgttatcc gctcacaatt ccacaataac ttcgtatagc atacattata cgaagttata
     1021 cccagctttc ttgtacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa
     1081 cgaacaggtc actatcagtc aaaataaaat cattatttgc catccagctg atatccccta
     1141 tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa
     1201 tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc
     1261 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc
     1321 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     1381 cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt
     1441 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     1501 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     1561 tgcatggtta ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata
     1621 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     1681 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     1741 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     1801 gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt
     1861 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     1921 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     1981 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     2041 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc
     2101 agaattggtt aattggttgt aacactggca gagcattacg ctgacttgac gggacggcgc
     2161 aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc
     2221 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2281 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2341 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     2401 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2461 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2521 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2581 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2641 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2701 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2761 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2821 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     2881 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     2941 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3001 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     3061 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     3121 taatacgcgt accgctagcc aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg
     3181 atggccttct gcttagtttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct
     3241 ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg gatttgtcct actcaggaga
     3301 gcgttcaccg acaaacaaca gataaaacga aaggcccagt cttccgactg agcctttcgt
     3361 tttatttgat gcctggcagt tccctactct cgcgttaacg ctagcatgga tgttttccca
     3421 gtcacgacgt t
//
