LOCUS       sequence-446908-        3299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A Gateway compatible middle entry clone containing a rtTA-3rd 
            generation transactivator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3299)
  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 3299)
  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 EFS-rtTA/rtTA3 (JDW 1230)
FEATURES             Location/Qualifiers
     source          1..3299
                     /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             669..1415
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     protein_bind    complement(1424..1523)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1540..1559)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1541..1559)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1564..1580)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1564..1580)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1682..1701)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1693..2502
                     /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(1760..1779)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2649..3237
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3138..3157
                     /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 agcaggctat gtctaggctg gacaagagca aagtcataaa ctctgctctg gaattactca
      721 atggagtcgg tatcgaaggc ctgacgacaa ggaaactcgc tcaaaagctg ggagttgagc
      781 agcctaccct gtactggcac gtgaagaaca agcgggccct gctcgatgcc ctgccaatcg
      841 agatgctgga caggcatcat acccactcct gccccctgga aggcgagtca tggcaagact
      901 ttctgcggaa caacgccaag tcataccgct gtgctctcct ctcacatcgc gacggggcta
      961 aagtgcatct cggcacccgc ccaacagaga aacagtacga aaccctggaa aatcagctcg
     1021 cgttcctgtg tcagcaaggc ttctccctgg agaacgcact gtacgctctg tccgccgtgg
     1081 gccactttac actgggctgc gtattggagg aacaggagca tcaagtagca aaagaggaaa
     1141 gagagacacc taccaccgat tctatgcccc cacttctgaa acaagcaatt gagctgttcg
     1201 accggcaggg agccgaacct gccttccttt tcggcctgga actaatcata tgtggcctgg
     1261 agaaacagct aaagtgcgaa agcggcgggc cgaccgacgc ccttgacgat tttgacttag
     1321 acatgctccc agccgatgcc cttgacgact ttgaccttga tatgctgcct gctgacgctc
     1381 ttgacgattt tgaccttgac atgctccccg ggtaatgata ggaattcagc tttcttgtac
     1441 aaagttggca ttataagaaa gcattgctta tcaatttgtt gcaacgaaca ggtcactatc
     1501 agtcaaaata aaatcattat ttgccatcca gctgatatcc cctatagtga gtcgtattac
     1561 atggtcatag ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg atgttacatt
     1621 gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat
     1681 acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac
     1741 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     1801 acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     1861 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     1921 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     1981 actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     2041 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     2101 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     2161 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     2221 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     2281 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     2341 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     2401 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     2461 aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg
     2521 ttgtaacact ggcagagcat tacgctgact tgacgggacg gcgcaagctc atgaccaaaa
     2581 tcccttaacg tgagttacgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2641 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2701 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2761 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     2821 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2881 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2941 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3001 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3061 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3121 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3181 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3241 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtt
//
