LOCUS       sequence-330532-        4086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway attB4/attB1r entry clone for mammalian expression using the 
            human EF1alpha promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4086)
  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 4086)
  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-E15: attB4-EF1p>-attB1r
FEATURES             Location/Qualifiers
     source          1..4086
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..45
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     37..54
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     38..54
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    110..205
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        213..1391
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          444..1382
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1339..1359
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1392..1516
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1588..1607)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1589..1607)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1612..1628)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1612..1628)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2053..2844
                     /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      2937..3525
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3426..3445
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3679..3696
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3855..3882)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3974..4060)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttaacgctag catggatgtt ttcccagtca cgacgttgta aaacgacggc cagtcttaaa
       61 attcgagctc ggtccgatcg ggcccgcgtt aacgctacca tggagctcca aataatgatt
      121 ttattttgac tgatagtgac ctgttcgttg caacaaattg ataagcaatg cttttttata
      181 atgccaactt tgtatagaaa agttggccgt gaggctccgg tgcccgtcag tgggcagagc
      241 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct
      301 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
      361 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
      421 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct
      481 ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctgcag tacgtgattc
      541 ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag
      601 ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa
      661 tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt
      721 tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag
      781 atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc
      841 agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt
      901 agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc
      961 cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc
     1021 ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg
     1081 agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca
     1141 cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg agtacgtcgt
     1201 ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag tgggtggaga
     1261 ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt
     1321 ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc
     1381 aggtgtcgtg aacaactttg tacaaaaaag ttgaacgaga aacgtaaaat gatataaata
     1441 tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac
     1501 atatgcagtc actatgaacc aactacttag atggtattag tgacctgtag aattcgagct
     1561 ctagagctgc agggcggccg cgatatcccc tatagtgagt cgtattacat ggtcatagct
     1621 gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagatcgg
     1681 tacccgggga tcctctagac cagccaggac agaaatgcct cgacttcgct gctacccaag
     1741 gttgccgggt gacgcacacc gtggaaacgg atgaaggcac gaacccagtg gacataagcc
     1801 tgttcggttc gtaagctgta atgcaagtag cgtatgcgct cacgcaactg gtccagaacc
     1861 ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt
     1921 ttttttgggg tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt
     1981 cgatgtttga tgttatggag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca
     2041 aagttaaaca ttatgaggga agcggtgatc gccgaagtat cgactcaact atcagaggta
     2101 gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt gtacggctcc
     2161 gcagtggatg gcggcctgaa gccacacagt gatattgatt tgctggttac ggtgaccgta
     2221 aggcttgatg aaacaacgcg gcgagctttg atcaacgacc ttttggaaac ttcggcttcc
     2281 cctggagaga gcgagattct ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc
     2341 attccgtggc gttatccagc taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac
     2401 attcttgcag gtatcttcga gccagccacg atcgacattg atctggctat cttgctgaca
     2461 aaagcaagag aacatagcgt tgccttggta ggtccagcgg cggaggaact ctttgatccg
     2521 gttcctgaac aggatctatt tgaggcgcta aatgaaacct taacgctatg gaactcgccg
     2581 cccgactggg ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc
     2641 gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat ggagcgcctg
     2701 ccggcccagt atcagcccgt catacttgaa gctagacagg cttatcttgg acaagaagaa
     2761 gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg tccactacgt gaaaggcgag
     2821 atcaccaagg tagtcggcaa ataaccctcg agccacccat gaccaaaatc ccttaacgtg
     2881 agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2941 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3001 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3061 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     3121 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3181 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3241 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3301 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3361 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3421 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3481 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3541 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3601 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3661 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     3721 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     3781 gactggaaag cgggcagtga gcgcaacgca attaatacgc gtaccgctag ccaggaagag
     3841 tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt tgatgcctgg
     3901 cagtttatgg cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca acgttcaaat
     3961 ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac
     4021 gaaaggccca gtcttccgac tgagcctttc gttttatttg atgcctggca gttccctact
     4081 ctcgcg
//
