LOCUS       sequence-11533-e        6830 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6830)
  AUTHORS   Frokjaer-Jensen C, Davis MW, Hollopeter G, Taylor J, Harris TW, Nix 
            P, Lofgren R, Prestgard-Duke M, Bastiani M, Moerman DG, Jorgensen EM
  TITLE     Targeted gene deletions in C. elegans using transposon excision.
  JOURNAL   Nat Methods. 2010 Jun;7(6):451-3. doi: 10.1038/nmeth.1454. Epub 2010
            Apr 25.
  PUBMED    20418868
REFERENCE   2  (bases 1 to 6830)
  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: 
            "10.1038/nmeth.1454"; journalName: "Nat Methods"; date: "2010-06"; 
            volume: "7"; issue: "6"; pages: "451-3"
COMMENT     Sequence Label: pCFJ66
FEATURES             Location/Qualifiers
     source          1..6830
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..18
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2..18
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    34..133
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(282..293)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2312..2334
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3301..3321)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     4069..4097
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     protein_bind    complement(4420..4519)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4536..4555)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4537..4555)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4560..4576)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4560..4576)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4678..4697)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4689..5498
                     /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(4756..4775)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5645..6233
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6134..6153
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6387..6404
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6563..6590)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(6682..6768)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(6817..6830,1..9)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tgtaaaacga cggccagtct taagctcggg ccccaaataa tgattttatt ttgactgata
       61 gtgacctgtt cgttgcaaca aattgatgag caatgctttt ttataatgcc aactttgtac
      121 aaaaaagcag gctctttgag ccaatttatc caagtccttg taaaaagtat tcgaaaattg
      181 ttaacggata aaatgtttat tataatatca aaaacaattt gtcagttgac cactttttga
      241 tatatttgac agaaaccggg atgaattggc tcaaagttag ggcgcccttc tattacaggg
      301 tttctgataa caaaccggtt attaactccc aacaagggat gatttcaatt catcatgctc
      361 aaattgaccc aaattaagtt acatgacaaa ttcatcgccc ttttcaactc ttcttggctc
      421 atcatctgtt attctgttcc tattctgtct gcaccctata cccctttgca tactctctcg
      481 tcattccttt cttggatagt gtcttcttct ccccagctct gctacttcta tgacttgcgc
      541 ccgctgcttt tccgctcgtt cctctcctct ctgacgtctt cttctgcttc ttttctcctt
      601 cctttttccc ttctcaatct tcctcttttt cccatttctg tcaatcattc gaagaagaag
      661 aagaagaaga ccctcattca tttcattttt ttttctgtcg gtgtgtgctg ctcggttaag
      721 agtgagctct ctattccacg tcttcttctt tttcttcttc gattcgaatc aatcactcca
      781 ccaaaacgca ttcgtttttg ggattcaccc cgcggttcgc aataggtttc tttttcaaat
      841 tattagcgtt ataaatagaa aatggtggag tttcaataaa aatgataatt tacaaagtgt
      901 attttgatta catgtactca aaaaggttga aatttttcat accagttttc cggaaatcca
      961 tctgatatca ttatcgtatt tttcttttta aaaatgtttt caaaaaaaaa caaaatatag
     1021 ctggttattt ggcaccctct aattaccatt ttcctgtcac accacactct tttccttctt
     1081 ccacttcttt ccgttttcag ccgcttccaa ccaaaccgat atgaaagccg agcaacaaca
     1141 atcgattcca cccggctcgg cgaccttccc gtcgcaggtg agactcagaa aactagagaa
     1201 accgctcaac taactcttga tatccgattt cattcttttc tcttttcttt ttgttgaact
     1261 tttccatatt tccagatgcc acggccacca ccaagcaccg aacaaggaat cacaacggaa
     1321 tcggagcttg cgaagaaagc tcaaatcact ccgaacgacg ttttagcact tccgggaatc
     1381 actcaaggta tcttgctccc tttggatttc agtttaaaac ataaatttag gattcttatg
     1441 ctccccatct gcgaacatct ataacatcga gttcaccaag ttccaaatcc gtgatctgga
     1501 cactgagcaa gtgcttttcg agatcgccaa accggagaac gatcaggaga atgatgagtc
     1561 gccacaggag tcggcaagat acgtgcgtta tagatttgct ccaaactttt tgaaactcaa
     1621 aacggtcgga gcaactgtgg aattcaaagt aggagacatc ccaatccatc atttccgaat
     1681 gatcgaacgt cacttcttca aagatcgcct tctgaagtgt tttgactttg aattcggatt
     1741 ctgtattccg aattcacgaa acaactgtga acatatctat gagttccctc aactctctca
     1801 acaactcagt gagtcattat tctaaaaagt acaattcaaa agactaatct ctttttcagt
     1861 ggacgacatg atcaacaatc caaacgagac tcgttctgac agcttctatt tcgtcgataa
     1921 caaactcgtc atgcacaaca aagccgacta ctcatatgat gcataaatat ttaatacaaa
     1981 aatgttctgg ataattattc tgtcgaatag aaaaaaaact ccaaatgtga ttaaattcca
     2041 ataattcctg tctagtttgt tccttcccct tccccttctc atgttcaatg cattcctaag
     2101 cttttcagtt ccccccttgt ttctatattt tttcgctgtc ctgtcacact cgctaaaaac
     2161 actaatcaca cggaaatctg ttttcaataa aaactccaac tttaactcat tttcaatttc
     2221 aactgaaaga ttttttcatt agagaatgtc tagaactagg cggccgcccg ggcagatctg
     2281 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     2341 ccgtcatcac cgaaacgcgc gagacgaaag ggcccgtacg gccgactagt aggaaacagt
     2401 tatgtttggt atattgggaa tgtattctgt catttaaggc aaaaacccca tagacactac
     2461 tccactttca aattatcaac ttctactcac cttccactga gcctcaaacc caaaccttct
     2521 tccgatcttt cttgcatcgt gctcatcaat acttgtgatt ttagcaagaa aaacgccgtc
     2581 ctcgaaatct tgacaaacaa taggggaaac cccaaaaagc aaaagcagga aaattaattt
     2641 cataaacttt cacttgattg aaaaattcca aaaatactcc aaaacttttt cacgatgagc
     2701 atgatttgac gtcatgagag gcccagacgt gcgaagaaat aaaaattgcg gtcataaact
     2761 gaaacgtaac atatgataag gtattttgtg tgcggttttt tctatgatgt ttcaaaaaaa
     2821 tttatcagaa gtaaaaaaca taagaagtgt acacagaaca ttgtgtagat tttgggatag
     2881 aattattatt aatgggggga gggagcacaa tttttttcga ctttttatta cgaattaatt
     2941 ctatttttga atctacacaa tttcattgtt agaggtgact taaaagaagc taaaaaacaa
     3001 agaaattaag agaagttttt tgataataac aaaaataggg ggtgggagca cagggagaaa
     3061 gagcatgtag ggatgttgaa gagtaattgg acttagaagt cagaggcacg ggcgcgagat
     3121 gctgaaaatt taaatattac tcgttagtta gtagaactca gtttaaactt actctgatga
     3181 cagcggcccc tattattttt gacaccagac aagttggtaa tggtagcgac cggcgctcag
     3241 ttggaattct acgaatgcta tttgtatagt tcatccatgc catgtgtaat cccagcagct
     3301 gttacaaact caagaaggac catgtggtct ctcttttcgt tgggatcttt cgaaagggca
     3361 gattgtgtgg acaggtaatg gttgtctggt aaaaggacag ggccatcgcc aattggagta
     3421 ttttgttgat aatggtctgc tagttgaacg cttccatctt caatgttgtg tctaattttg
     3481 aagttctgaa aatttaaata taatccgtta gttagtaagt ccaagtttaa acttacaact
     3541 ttgattccat tcttttgttt gtctgccatg atgtatacat tgtgtgagtt atagttgtat
     3601 tccaatttgt gtccaagaat gtttccatct tctttaaaat caataccttt taactcgatt
     3661 ctattaacaa gggtatcacc ttcaaacttg acttcagcac ctgaaaattt aaatatgtat
     3721 ggttagttag taccgaactg tttaaactta cgtgtcttgt agttcccgtc atctttgaaa
     3781 aatatagttc tttcctgtac ataaccttcg ggcatggcac tcttgaaaaa gtcatgccgt
     3841 ttcatatgat ctgggtatct cgagaagcat tgaacaccat aacagaaagt agtgacaagt
     3901 gttggctgaa aatttaaata atcagggtta gttagtatat atatgtttaa acttacccat
     3961 ggaacaggta gttttccagt agtgcaaata aatttaaggg taagttttcc gtatgttgca
     4021 tcaccttcac cctctccact gacagaaaat ttgtgcccat taacatcacc atctaattca
     4081 acaagaattg ggacaactcc agtgaaaagt tcttctcctt tactcatttt ttctaccggt
     4141 accctcaagg gtcctcctga aaatgttcta tgttatgtta gtatcattcg aaacatacct
     4201 ttgggtcctt tggccaatcc cggggatcct gtcgcaacca tattgtgagc ccaatgaagt
     4261 aaaatttcat gctagttgcc agaatatagt ggtaatatga tatgcacgta gaataaacaa
     4321 attgttgact gatagtcatt tccgtggggt tttggaaatg tttagaataa gtctccaaat
     4381 attgcgtgaa ggatgagaaa tgagggaaac tgcgtgtcta cccagctttc ttgtacaaag
     4441 ttggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc
     4501 aaaataaaat cattatttgc catccagctg atatccccta tagtgagtcg tattacatgg
     4561 tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac
     4621 aagataaaat aatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     4681 ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg
     4741 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     4801 tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     4861 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     4921 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     4981 cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     5041 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     5101 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
     5161 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     5221 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     5281 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     5341 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     5401 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     5461 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt
     5521 aacactggca gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc
     5581 ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5641 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5701 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5761 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     5821 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5881 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5941 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6001 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6061 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6121 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6181 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6241 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     6301 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     6361 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     6421 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     6481 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatacgcgt accgctagcc
     6541 aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttagtttg
     6601 atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac
     6661 gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca
     6721 gataaaacga aaggcccagt cttccgactg agcctttcgt tttatttgat gcctggcagt
     6781 tccctactct cgcgttaacg ctagcatgga tgttttccca gtcacgacgt
//
