LOCUS       sequence-78690-e        4249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry vector with attL5 and attL2 sites flanking a CmR and 
            ccdB gene. In our hands, the multisite reaction still proceeds 
            efficiently. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4249)
  AUTHORS   Baltes NJ, Gil-Humanes J, Cermak T, Atkins PA, Voytas DF
  TITLE     DNA Replicons for Plant Genome Engineering.
  JOURNAL   Plant Cell. 2014 Jan;26(1):151-63. doi: 10.1105/tpc.113.119792
  PUBMED    24443519
REFERENCE   2  (bases 1 to 4249)
  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"; doi: "10.1105/tpc.113"; 
            journalName: "Plant Cell"; date: "2014-01"; volume: "26"; issue: 
            "1"; pages: "151-63"
COMMENT     Sequence Label: pNJB80
FEATURES             Location/Qualifiers
     source          1..4249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(31..36,37..54,55..61)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             115..795
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(181..200)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1115..1420
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1299..1318
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1462..1561)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1578..1597)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1579..1597)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1602..1618)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1602..1618)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2049..2840
                     /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      2933..3521
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3422..3441
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3675..3692
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3851..3878)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3970..4056)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4105..4127
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4119..4136
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4120..4136
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4152..4247
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gagctcgcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat
       61 gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag
      121 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
      181 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
      241 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
      301 cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg
      361 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
      421 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
      481 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
      541 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
      601 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
      661 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
      721 agaatgctta atgaattaca acagtactgc gatgagtggc aggcggggcg taatctagag
      781 gatccggctt actaaaagcc agataacagt atgcgtattt gcgcgctgat ttttgcggta
      841 taagaatata tactgatatg tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca
      901 gcgtattaca gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc
      961 aatatctccg gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa
     1021 cgctggaaag cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac
     1081 ggctcttttg ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa
     1141 aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg
     1201 gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag tcccccgtga
     1261 actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc
     1321 cagtgtgccg gtctccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga
     1381 catcaaaaac gccattaacc tgatgttctg gggaatataa atgtcaggct cccttataca
     1441 cagccagtct gcaggtcgac gacccagctt tcttgtacaa agttggcatt ataaaaaata
     1501 attgctcatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt
     1561 gccatccagc tgatatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc
     1621 agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat
     1681 catgcctcct ctggaccagc caggacagaa atgcctcgac ttcgctgcta cccaaggttg
     1741 ccgggtgacg cacaccgtgg aaacggatga aggcacgaac ccagtggaca taagcctgtt
     1801 cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg caactggtcc agaaccttga
     1861 ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat ggcttgttat gactgttttt
     1921 ttggggtaca gtctatgcct cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat
     1981 gtttgatgtt atggagcagc aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt
     2041 taaacatcat gagggaagcg gtgatcgccg aagtatcgac tcaactatca gaggtagttg
     2101 gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt acatttgtac ggctccgcag
     2161 tggatggcgg cctgaagcca cacagtgata ttgatttgct ggttacggtg accgtaaggc
     2221 ttgatgaaac aacgcggcga gctttgatca acgacctttt ggaaacttcg gcttcccctg
     2281 gagagagcga gattctccgc gctgtagaag tcaccattgt tgtgcacgac gacatcattc
     2341 cgtggcgtta tccagctaag cgcgaactgc aatttggaga atggcagcgc aatgacattc
     2401 ttgcaggtat cttcgagcca gccacgatcg acattgatct ggctatcttg ctgacaaaag
     2461 caagagaaca tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt gatccggttc
     2521 ctgaacagga tctatttgag gcgctaaatg aaaccttaac gctatggaac tcgccgcccg
     2581 actgggctgg cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag
     2641 taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg
     2701 cccagtatca gcccgtcata cttgaagcta gacaggctta tcttggacaa gaagaagatc
     2761 gcttggcctc gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa ggcgagatca
     2821 ccaaggtagt cggcaaataa ccctcgagcc acccatgacc aaaatccctt aacgtgagtt
     2881 acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2941 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3001 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3061 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3121 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3181 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3241 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3301 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3361 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3421 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3481 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3541 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3601 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3661 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     3721 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     3781 ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac cgcgagccag gaagagtttg
     3841 tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat gcctggcagt
     3901 ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc
     3961 tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa
     4021 ggcccagtct tccgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg
     4081 cgttaacgct tgcatggatg ttttcccagt cacgacgttg taaaacgacg gccagtctta
     4141 agctcgggcc caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat
     4201 tgatgagcaa tgctttttta taatgccaac tttgtataca aaagttgcc
//
