LOCUS       sequence-333333-        3278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB4/attB1r entry clone for inducible mammalian expression 
            using the TRE3G promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3278)
  AUTHORS   Wall VE, Garvey LA, Mehalko JL, Procter LV, Esposito D
  TITLE     Combinatorial assembly of clone libraries using site-specific 
            recombination.
  JOURNAL   Methods Mol Biol. 2014;1116:193-208. doi: 
            10.1007/978-1-62703-764-8_14.
  PUBMED    24395366
REFERENCE   2  (bases 1 to 3278)
  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: "Methods Mol
            Biol."; date: "2014"; volume: "1116:193-208. doi"; pages: " 
            10.1007/978-1-62703-764-8_14"
COMMENT     Sequence Label: C413-E31: attB4-TRE3Gp>-attB1r
FEATURES             Location/Qualifiers
     source          1..3278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(52..138)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     187..209
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     201..218
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     202..218
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    274..369
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        369..747
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    377..395
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    413..431
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    449..467
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    485..503
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    521..539
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    557..575
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    593..611
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     656..680
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    748..872
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(944..963)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(945..963)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(968..984)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(968..984)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1409..2200
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      2293..2881
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2782..2801
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3035..3052
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3211..3238)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg gatttgtcct
       61 actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt cttccgactg
      121 agcctttcgt tttatttgat gcctggcagt tccctactct cgcgttaacg ctagcatgga
      181 tgttttccca gtcacgacgt tgtaaaacga cggccagtct taaaattcga gctcggtccg
      241 atcgggcccg cgttaacgct accatggagc tccaaataat gattttattt tgactgatag
      301 tgacctgttc gttgcaacaa attgataagc aatgcttttt tataatgcca actttgtata
      361 gaaaagttgg ctttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
      421 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      481 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      541 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      601 agtgatagag aacgtataag ctttaggcgt gtacggtggg cgcctataaa agcagagctc
      661 gtttagtgaa ccgtcagatc gcctggagca attccacaac acttttgtct tataccaact
      721 ttccgtacca cttcctaccc tcgtaaaaca actttgtaca aaaaagttga acgagaaacg
      781 taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa
      841 tactgtaaaa cacaacatat gcagtcacta tgaaccaact acttagatgg tattagtgac
      901 ctgtagaatt cgagctctag agctgcaggg cggccgcgat atcccctata gtgagtcgta
      961 ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta
     1021 cattgcacaa gatcggtacc cggggatcct ctagaccagc caggacagaa atgcctcgac
     1081 ttcgctgcta cccaaggttg ccgggtgacg cacaccgtgg aaacggatga aggcacgaac
     1141 ccagtggaca taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg
     1201 caactggtcc agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat
     1261 ggcttgttat gactgttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc
     1321 gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg
     1381 cagtcgccct aaaacaaagt taaacattat gagggaagcg gtgatcgccg aagtatcgac
     1441 tcaactatca gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt
     1501 acatttgtac ggctccgcag tggatggcgg cctgaagcca cacagtgata ttgatttgct
     1561 ggttacggtg accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt
     1621 ggaaacttcg gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt
     1681 tgtgcacgac gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga
     1741 atggcagcgc aatgacattc ttgcaggtat cttcgagcca gccacgatcg acattgatct
     1801 ggctatcttg ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc cagcggcgga
     1861 ggaactcttt gatccggttc ctgaacagga tctatttgag gcgctaaatg aaaccttaac
     1921 gctatggaac tcgccgcccg actgggctgg cgatgagcga aatgtagtgc ttacgttgtc
     1981 ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg
     2041 ggcaatggag cgcctgccgg cccagtatca gcccgtcata cttgaagcta gacaggctta
     2101 tcttggacaa gaagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgtcca
     2161 ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa ccctcgagcc acccatgacc
     2221 aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     2281 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2341 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2401 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     2461 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2521 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2581 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     2641 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     2701 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     2761 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2821 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     2881 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     2941 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3001 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3061 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     3121 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac
     3181 cgctagccag gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc
     3241 ttagtttgat gcctggcagt ttatggcggg cgtcctgc
//
