LOCUS       sequence-42405-e        5970 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 5970)
  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 5970)
  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: pBCN44-R4R3
FEATURES             Location/Qualifiers
     source          1..5970
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             782..1576
                     /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             782..1576
                     /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(836..855)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1446..1465
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2151..2173
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2211..2229)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2297..2401
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2402..2470,2471..3262)
                     /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(2620..2639)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3433..4021
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3922..3941
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4147..4169
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4166..4182
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4166..4182
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    4202..4325
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(4366..4671)
                     /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(4468..4487)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(4991..5674)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     5589..5608
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(5728..5734,5735..5752,5753..5758))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(5783..5906)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5914..5931)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5914..5930)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5923..5945)
                     /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 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
      841 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
      901 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
      961 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     1021 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     1081 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     1141 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     1201 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     1261 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     1321 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     1381 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     1441 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     1501 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     1561 tgacgagttc ttctgagcta gccggccata caagtaatcc ggatgatcga cgccaacgtc
     1621 gttgaatttt caaattttaa atactgaata tttgtttttt ttcctattat ttatttattc
     1681 tctttgtgtt ttttttcttg ctttctaaaa aattaattca atccaaatct aaacattttt
     1741 ttttctcttt ccgtctccca attcgtattc cgctcctctc atctgaacac aatgtgcaag
     1801 tttatttatc ttctcgcttt catttcatta ggacgtgggg ggaattggtg gaagggggaa
     1861 acacacaaaa ggatgatgga aatgaaataa ggacacacaa tatgcaacaa cattcaattc
     1921 agaaatatgg aggaaggttt aaaagaaaac ataaaaatat atagaggagg aaggaaaact
     1981 agtccaagat ctcagtggtc ctaggtgagc tcccatatgg tgcactctca gtgcaatctg
     2041 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     2101 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     2161 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     2221 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     2281 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2341 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2401 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2461 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     2521 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2581 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     2641 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2701 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2761 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     2821 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     2881 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     2941 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3001 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3061 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     3121 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     3181 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     3241 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     3301 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     3361 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3421 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3481 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3541 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     3601 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3661 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3721 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3781 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3841 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3901 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3961 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4021 aaacgccagc aacgcggcct ttttacggtt cctggtcttt tgctggcctt tgctcacatg
     4081 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4141 gataccagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcta
     4201 tcaactttgt atagaaaagt tgaacgagaa acgtaaaatg atataaatat caatatatta
     4261 aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca
     4321 ctatggtcga cctgcagact ggctgtgtat aagggagcct gacatttata ttccccagaa
     4381 catcaggtta atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc
     4441 cccgataacg gagaccggca cactggccat atcggtggtc atcatgcgcc agctttcatc
     4501 cccgatatgc accaccgggt aaagttcacg ggagacttta tctgacagca gacgtgcact
     4561 ggccaggggg atcaccatcc gtcgcccggg cgtgtcaata atatcactct gtacatccac
     4621 aaacagacga taacggctct ctcttttata ggtgtaaacc ttaaactgca tttcaccagc
     4681 ccctgttctc gtcagcaaaa gagccgttca tttcaataaa ccgggcgacc tcagccatcc
     4741 cttcctgatt ttccgctttc cagcgttcgg cacgcagacg acgggcttca ttctgcatgg
     4801 ttgtgcttac cagaccggag atattgacat catatatgcc ttgagcaact gatagctgtc
     4861 gctgtcaact gtcactgtaa tacgctgctt catagcatac ctctttttga catacttcgg
     4921 gtatacatat cagtatatat tcttataccg caaaaatcag cgcgcaaata cgcatactgt
     4981 tatctggctt ttagtaagcc ggatccacgc ggcgtttacg ccccccctgc cactcatcgc
     5041 agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat
     5101 gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg
     5161 tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac
     5221 tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg
     5281 ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat
     5341 cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt
     5401 aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc atacggaatt
     5461 ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct
     5521 tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg
     5581 tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat
     5641 caacggtggt atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc
     5701 tcgacggatc ctaactcaaa atccacacat tatacgagcc ggaagcataa agtgtaaagc
     5761 ctggggtgcc taatgcggcc gccatagtga ctggatatgt tgtgttttac agtattatgt
     5821 agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc
     5881 tcgttcaact ttattataca tagttgataa ttcactggcc gtcgttttac aacgtcgtga
     5941 ctgggaaaac cctggcgtta cccaacttaa
//
