LOCUS       Exported                9104 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pBCN40-R4R3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9104)
  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 9104)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9104
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(3023..3042)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3621..3641
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3916..4710
                     /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(3970..3989)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4580..4599
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5285..5307
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5345..5363)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5431..5535
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5536..6396
                     /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(5754..5773)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6567..7155
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7056..7075
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7281..7303
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7300..7316
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7300..7316
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    7335..7459
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(7500..7805)
                     /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(7602..7621)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(8155..8808)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG"
     primer_bind     8723..8742
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(8862..8892)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(8917..9040)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(9048..9065)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9048..9064)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9057..9079)
                     /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 gccccatttt atatctgagt
      181 agtatccttt gctttaaatg tccataaaac taattttata atcaataaaa caacgtttgt
      241 aaatcaactg agtttacaag tagagacatt gagggatact ttcactatgc taaagtgata
      301 atcgaccaaa taataacttc actttggtat ttattcctgt cttataaatg ttatgtatga
      361 attaaattca tatgcatatg gctcactctg acaattttta ataatcttcc agatcaatat
      421 tgactaccga tgcgggtggt cttttgcttt gaattctgct gaactttaca ccccgaacag
      481 caatgtgtgc ttcagcctaa aaaaaagtaa gtgtgttaat cagtgccccc gattcttcat
      541 tttttgcccc tctctcccgt ttcgtcggca aaagaagaga aaataaagat aagtctcaag
      601 ataggttggt aatcgctaaa gtggttgtgt ggataagagt agcaaaatgg caggaagagc
      661 actttgcgcg cacacactgt actcattgtt ctggataaaa ttctctcgtt gtttgccgtc
      721 ggatgtctgc ctctctgcat tgagccggct tcttcactat ctttagttaa cctaaaatgc
      781 cgtttctttt ctcgtatccc cactatcccg ttgaggttct ctgctctctt cgctccctac
      841 cgccagcgag caactatccg tgggggcgcc ttgctcggaa gatggggggg aagaaagaag
      901 atttttgcta tttgcacttg agaaagagac ttttcctgcg tcgatggtta gagaacagtg
      961 tgcagacact tttcagctac ctagaattac aattggatat ccccgcctcc caatccaccc
     1021 acccagggaa aaagaagggc tcgccgaaaa tcaaagttat ctccaggctc gcgcatccca
     1081 ccgagcggtt gacttctctc caccactttt cattttaacc ctcgatcgtc agacacagaa
     1141 atggtctcaa agggtgaaga agataacatg gcaattatta aagagtttat gcgtttcaag
     1201 gtgcatatgg agggatctgt caatgggcat gagtttgaaa ttgaaggtga aggagaaggc
     1261 cgaccatatg agggaacaca aaccgcaaaa ctaaaggtaa gtttaaacat atatatacta
     1321 actaaccctg attatttaaa ttttcaggta actaaaggcg gaccattacc attcgcctgg
     1381 gacatcctct ctccacagtt catgtatgga agtaaagctt atgttaaaca tccggcagat
     1441 ataccagatt atttgaaact ttcattcccg gagggtttta agtgggaacg cgtaatgaat
     1501 tttgaagacg gaggagttgt tacagtgacg caagactcaa ggtaagttta aacagttcgg
     1561 tactaactaa ccatacatat ttaaattttc agcctccaag atggagaatt tatttataaa
     1621 gtcaaacttc gaggaacgaa tttcccctcg gatggacctg ttatgcagaa gaagactatg
     1681 ggatgggaag cttcaagtga aagaatgtac cctgaagacg gtgctcttaa gggagagatt
     1741 aaacaacgtc ttaaattgaa agatggagga cattacgatg ctgaggtaag tttaaacatg
     1801 attttactaa ctaactaatc tgatttaaat tttcaggtga agacaactta caaagccaaa
     1861 aaaccagttc agctgccagg agcgtacaat gttaatatta aactggatat cacctcccac
     1921 aacgaggatt acactatcgt tgagcaatat gaaagagctg aagggcggca ctcgacaggt
     1981 ggcatggatg aattgtataa gtagatcaac tgctctaatt ccatctcgtt tctctctttt
     2041 ctttcaatct ctttacccgt ttctcttccc ctacgtcccc caaacctacc actgcacagt
     2101 aacacataca cattcaacgt gtcacaataa aagcccatca acaccttctc tgttgttttt
     2161 tttcttcaaa tgcatttttt tcactttgtt taataatttt tacgcatcgg atttgtagga
     2221 tagtggaaat ttgtccttca atgcttcaat tgattcgaca cttctcaata tttttgatca
     2281 ataaaccatt gattttctat ttagaagaag atcactgtaa ataacaccaa aaacatcaca
     2341 gcaaaaaaac tcccactcaa ttatattact gtttttattg aaactagaat aaatttgaag
     2401 ggcccccctc gaggggagaa aatgcggaga gaagaaccca gcatcttaat aaaattatat
     2461 aattttgaat aaaatcaaat taaaaataag attaattgaa acacaacaat agaatgcaga
     2521 aaattactca taagaaataa tttattttga aagaagttgt gtcgatttac gataaaaact
     2581 acacgcaaac gaggctccgc gtttacaccg tttggcaatg gagcgcgctt gctattagat
     2641 tttaggccag ctcaacgaaa gtttaattta tttttcttct ttttttccat ttttttgagt
     2701 aaaatatcga attttttgta ttaattctgt aaattttata atttgcaact attttggcgt
     2761 attctgcttg tttttatcag cgaattttta tgcatttcag tcaaaagttt gagcaggaaa
     2821 aatgacattt aggactttgt aaaattgtat tcttaaattt gcttgaaaac ctacgtgtac
     2881 aattgatttt attgaatttt tcttacgttt cccgttgtat cttattgtat ttaactttca
     2941 tcataaaata atttatttcc aggtaaaata tccgacgctc tcgtggatcc ccgggattgg
     3001 ctcagaaaaa aaggtaccga ccatgaccga gtacaagccc acggtgcgcc tcgccacccg
     3061 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     3121 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     3181 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc ggtaagttta aacatatata
     3241 tactaactaa ccctgattat ttaaattttc agcggacgac ggcgccgcgg tggcggtctg
     3301 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     3361 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     3421 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg acgtaagttt
     3481 aaacatgatt ttactaacta actaatctga tttaaatttt cagcaccagg gcaagggtct
     3541 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     3601 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     3661 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     3721 ctgatgagct agcgattact gaaatatgta aatattagaa gaatttgtgc gggattcttg
     3781 ccaaataaat tctcgaaata gttttcatat atttattgtt atgcgcgttc tctcgcttcg
     3841 atttatctct cttcctttac tctcgtgtat ttctaaactt tccccttctt atcagtattt
     3901 atttcagggt aaacgatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3961 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     4021 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     4081 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     4141 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     4201 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     4261 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     4321 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     4381 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     4441 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     4501 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     4561 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4621 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4681 ttctatcgcc ttcttgacga gttcttctga gctagccggc catacaagta atccggatga
     4741 tcgacgccaa cgtcgttgaa ttttcaaatt ttaaatactg aatatttgtt ttttttccta
     4801 ttatttattt attctctttg tgtttttttt cttgctttct aaaaaattaa ttcaatccaa
     4861 atctaaacat ttttttttct ctttccgtct cccaattcgt attccgctcc tctcatctga
     4921 acacaatgtg caagtttatt tatcttctcg ctttcatttc attaggacgt ggggggaatt
     4981 ggtggaaggg ggaaacacac aaaaggatga tggaaatgaa ataaggacac acaatatgca
     5041 acaacattca attcagaaat atggaggaag gtttaaaaga aaacataaaa atatatagag
     5101 gaggaaggaa aactagtcca agatctcagt ggtcctaggt gagctcccat atggtgcact
     5161 ctcagtgcaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     5221 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     5281 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     5341 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     5401 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5461 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5521 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5581 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5641 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5701 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5761 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5821 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5881 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5941 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6001 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6061 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6121 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6181 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6241 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6301 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6361 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6421 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6481 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6541 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6601 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6661 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6721 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6781 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6841 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6901 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6961 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7021 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7081 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7141 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggt cttttgctgg
     7201 cctttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     7261 cctttgagtg agctgatacc agcggataac aatttcacac aggaaacagc tatgaccatg
     7321 attacgccaa gctatcaact ttgtatagaa aagttgaacg agaaacgtaa aatgatataa
     7381 atatcaatat attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac
     7441 aacatatcca gtcactatgg tcgacctgca gactggctgt gtataaggga gcctgacatt
     7501 tatattcccc agaacatcag gttaatggcg tttttgatgt cattttcgcg gtggctgaga
     7561 tcagccactt cttccccgat aacggagacc ggcacactgg ccatatcggt ggtcatcatg
     7621 cgccagcttt catccccgat atgcaccacc gggtaaagtt cacgggagac tttatctgac
     7681 agcagacgtg cactggccag ggggatcacc atccgtcgcc cgggcgtgtc aataatatca
     7741 ctctgtacat ccacaaacag acgataacgg ctctctcttt tataggtgta aaccttaaac
     7801 tgcatttcac cagcccctgt tctcgtcagc aaaagagccg ttcatttcaa taaaccgggc
     7861 gacctcagcc atcccttcct gattttccgc tttccagcgt tcggcacgca gacgacgggc
     7921 ttcattctgc atggttgtgc ttaccagacc ggagatattg acatcatata tgccttgagc
     7981 aactgatagc tgtcgctgtc aactgtcact gtaatacgct gcttcatagc atacctcttt
     8041 ttgacatact tcgggtatac atatcagtat atattcttat accgcaaaaa tcagcgcgca
     8101 aatacgcata ctgttatctg gcttttagta agccggatcc acgcggcgtt tacgcccccc
     8161 ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc
     8221 acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata
     8281 atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc
     8341 aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc
     8401 tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag
     8461 aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc
     8521 atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat
     8581 tgccatacgg aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg
     8641 ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac
     8701 ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg
     8761 ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt
     8821 agctcctgaa aatctcgacg gatcctaact caaaatccac acattatacg agccggaagc
     8881 ataaagtgta aagcctgggg tgcctaatgc ggccgccata gtgactggat atgttgtgtt
     8941 ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata
     9001 tcattttacg tttctcgttc aactttatta tacatagttg ataattcact ggccgtcgtt
     9061 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaa
//
