LOCUS       sequence-42400-e        6863 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 6863)
  AUTHORS   Semple JI, Biondini L, Lehner B
  TITLE     Generating transgenic nematodes by bombardment and antibiotic 
            selection.
  JOURNAL   Nat Methods. 2012 Jan 30;9(2):118-9. doi: 10.1038/nmeth.1864.
  PUBMED    22290182
REFERENCE   2  (bases 1 to 6863)
  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"; 
            journalName: "Nat Methods"; date: "2012-01-30- 30"; volume: "9"; 
            issue: "2"; pages: "118-9"
COMMENT     Sequence Label: pBCN39-R4R3
FEATURES             Location/Qualifiers
     source          1..6863
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(782..801)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1380..1400
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1675..2469
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             1675..2469
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1729..1748)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2339..2358
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3044..3066
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3104..3122)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3190..3294
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3295..3363,3364..4155)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3513..3532)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4326..4914
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4815..4834
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5040..5062
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5059..5075
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5059..5075
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5095..5218
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(5259..5564)
                     /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
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(5361..5380)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(5884..6567)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     6482..6501
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(6621..6627,6628..6645,6646..6651))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(6676..6799)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6807..6824)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6807..6823)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6816..6838)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
       61 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
      121 ccttacgcat ctgtgcggta tttcacaccg catatgccgg gcccccctcg aggggagaaa
      181 atgcggagag aagaacccag catcttaata aaattatata attttgaata aaatcaaatt
      241 aaaaataaga ttaattgaaa cacaacaata gaatgcagaa aattactcat aagaaataat
      301 ttattttgaa agaagttgtg tcgatttacg ataaaaacta cacgcaaacg aggctccgcg
      361 tttacaccgt ttggcaatgg agcgcgcttg ctattagatt ttaggccagc tcaacgaaag
      421 tttaatttat ttttcttctt tttttccatt tttttgagta aaatatcgaa ttttttgtat
      481 taattctgta aattttataa tttgcaacta ttttggcgta ttctgcttgt ttttatcagc
      541 gaatttttat gcatttcagt caaaagtttg agcaggaaaa atgacattta ggactttgta
      601 aaattgtatt cttaaatttg cttgaaaacc tacgtgtaca attgatttta ttgaattttt
      661 cttacgtttc ccgttgtatc ttattgtatt taactttcat cataaaataa tttatttcca
      721 ggtaaaatat ccgacgctct cgtggatccc cgggattggc tcagaaaaaa aggtaccgac
      781 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
      841 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
      901 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
      961 catcggcaag gtgtgggtcg gtaagtttaa acatatatat actaactaac cctgattatt
     1021 taaattttca gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     1081 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     1141 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     1201 gttcctggcc accgtcggcg tctcgcccga cgtaagttta aacatgattt tactaactaa
     1261 ctaatctgat ttaaattttc agcaccaggg caagggtctg ggcagcgccg tcgtgctccc
     1321 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     1381 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     1441 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgatgagcta gcgattactg
     1501 aaatatgtaa atattagaag aatttgtgcg ggattcttgc caaataaatt ctcgaaatag
     1561 ttttcatata tttattgtta tgcgcgttct ctcgcttcga tttatctctc ttcctttact
     1621 ctcgtgtatt tctaaacttt ccccttctta tcagtattta tttcagggta aacgatgatt
     1681 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     1741 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     1801 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac
     1861 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     1921 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     1981 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     2041 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     2101 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     2161 caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag
     2221 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     2281 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     2341 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     2401 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     2461 ttcttctgag ctagccggcc atacaagtaa tccggatgat cgacgccaac gtcgttgaat
     2521 tttcaaattt taaatactga atatttgttt tttttcctat tatttattta ttctctttgt
     2581 gttttttttc ttgctttcta aaaaattaat tcaatccaaa tctaaacatt tttttttctc
     2641 tttccgtctc ccaattcgta ttccgctcct ctcatctgaa cacaatgtgc aagtttattt
     2701 atcttctcgc tttcatttca ttaggacgtg gggggaattg gtggaagggg gaaacacaca
     2761 aaaggatgat ggaaatgaaa taaggacaca caatatgcaa caacattcaa ttcagaaata
     2821 tggaggaagg tttaaaagaa aacataaaaa tatatagagg aggaaggaaa actagtccaa
     2881 gatctcagtg gtcctaggtg agctcccata tggtgcactc tcagtgcaat ctgctctgat
     2941 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     3001 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     3061 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     3121 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     3181 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     3241 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     3301 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     3361 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     3421 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     3481 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     3541 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     3601 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     3661 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     3721 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     3781 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     3841 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     3901 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     3961 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     4021 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     4081 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     4141 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     4201 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     4261 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4321 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4381 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4441 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     4501 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4561 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4621 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4681 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4741 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4801 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4861 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4921 agcaacgcgg cctttttacg gttcctggtc ttttgctggc ctttgctcac atgttctttc
     4981 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgatacca
     5041 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctatcaactt
     5101 tgtatagaaa agttgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga
     5161 ttttgcataa aaaacagact acataatact gtaaaacaca acatatccag tcactatggt
     5221 cgacctgcag actggctgtg tataagggag cctgacattt atattcccca gaacatcagg
     5281 ttaatggcgt ttttgatgtc attttcgcgg tggctgagat cagccacttc ttccccgata
     5341 acggagaccg gcacactggc catatcggtg gtcatcatgc gccagctttc atccccgata
     5401 tgcaccaccg ggtaaagttc acgggagact ttatctgaca gcagacgtgc actggccagg
     5461 gggatcacca tccgtcgccc gggcgtgtca ataatatcac tctgtacatc cacaaacaga
     5521 cgataacggc tctctctttt ataggtgtaa accttaaact gcatttcacc agcccctgtt
     5581 ctcgtcagca aaagagccgt tcatttcaat aaaccgggcg acctcagcca tcccttcctg
     5641 attttccgct ttccagcgtt cggcacgcag acgacgggct tcattctgca tggttgtgct
     5701 taccagaccg gagatattga catcatatat gccttgagca actgatagct gtcgctgtca
     5761 actgtcactg taatacgctg cttcatagca tacctctttt tgacatactt cgggtataca
     5821 tatcagtata tattcttata ccgcaaaaat cagcgcgcaa atacgcatac tgttatctgg
     5881 cttttagtaa gccggatcca cgcggcgttt acgccccccc tgccactcat cgcagtactg
     5941 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg
     6001 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac
     6061 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca
     6121 gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt
     6181 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg
     6241 gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg
     6301 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg
     6361 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt
     6421 ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg
     6481 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt
     6541 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgacgg
     6601 atcctaactc aaaatccaca cattatacga gccggaagca taaagtgtaa agcctggggt
     6661 gcctaatgcg gccgccatag tgactggata tgttgtgttt tacagtatta tgtagtctgt
     6721 tttttatgca aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca
     6781 actttattat acatagttga taattcactg gccgtcgttt tacaacgtcg tgactgggaa
     6841 aaccctggcg ttacccaact taa
//
