LOCUS       Exported                8121 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pBCN43-R4R3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8121)
  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 8121)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8121
                     /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"
     primer_bind     4302..4324
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4362..4380)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4448..4552
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4553..5413
                     /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(4771..4790)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5584..6172
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6073..6092
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6298..6320
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6317..6333
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6317..6333
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    6352..6476
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(6517..6822)
                     /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(6619..6638)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(7172..7825)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG"
     primer_bind     7740..7759
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7879..7909)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(7934..8057)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(8065..8082)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8065..8081)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8074..8096)
                     /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 agccggccat acaagtaatc cggatgatcg acgccaacgt cgttgaattt
     3781 tcaaatttta aatactgaat atttgttttt tttcctatta tttatttatt ctctttgtgt
     3841 tttttttctt gctttctaaa aaattaattc aatccaaatc taaacatttt tttttctctt
     3901 tccgtctccc aattcgtatt ccgctcctct catctgaaca caatgtgcaa gtttatttat
     3961 cttctcgctt tcatttcatt aggacgtggg gggaattggt ggaaggggga aacacacaaa
     4021 aggatgatgg aaatgaaata aggacacaca atatgcaaca acattcaatt cagaaatatg
     4081 gaggaaggtt taaaagaaaa cataaaaata tatagaggag gaaggaaaac tagtccaaga
     4141 tctcagtggt cctaggtgag ctcccatatg gtgcactctc agtgcaatct gctctgatgc
     4201 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4261 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4321 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     4381 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     4441 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     4501 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     4561 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     4621 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     4681 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     4741 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     4801 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     4861 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     4921 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     4981 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5041 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5101 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     5161 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     5221 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     5281 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     5341 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     5401 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     5461 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     5521 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5581 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5641 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5701 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     5761 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5821 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5881 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     5941 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6001 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6061 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6121 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6181 caacgcggcc tttttacggt tcctggtctt ttgctggcct ttgctcacat gttctttcct
     6241 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccagc
     6301 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct atcaactttg
     6361 tatagaaaag ttgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt
     6421 ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc actatggtcg
     6481 acctgcagac tggctgtgta taagggagcc tgacatttat attccccaga acatcaggtt
     6541 aatggcgttt ttgatgtcat tttcgcggtg gctgagatca gccacttctt ccccgataac
     6601 ggagaccggc acactggcca tatcggtggt catcatgcgc cagctttcat ccccgatatg
     6661 caccaccggg taaagttcac gggagacttt atctgacagc agacgtgcac tggccagggg
     6721 gatcaccatc cgtcgcccgg gcgtgtcaat aatatcactc tgtacatcca caaacagacg
     6781 ataacggctc tctcttttat aggtgtaaac cttaaactgc atttcaccag cccctgttct
     6841 cgtcagcaaa agagccgttc atttcaataa accgggcgac ctcagccatc ccttcctgat
     6901 tttccgcttt ccagcgttcg gcacgcagac gacgggcttc attctgcatg gttgtgctta
     6961 ccagaccgga gatattgaca tcatatatgc cttgagcaac tgatagctgt cgctgtcaac
     7021 tgtcactgta atacgctgct tcatagcata cctctttttg acatacttcg ggtatacata
     7081 tcagtatata ttcttatacc gcaaaaatca gcgcgcaaat acgcatactg ttatctggct
     7141 tttagtaagc cggatccacg cggcgtttac gccccccctg ccactcatcg cagtactgtt
     7201 gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa
     7261 tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg
     7321 gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg
     7381 gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt
     7441 caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt
     7501 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt
     7561 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag
     7621 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct
     7681 ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag
     7741 caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg
     7801 tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat ctcgacggat
     7861 cctaactcaa aatccacaca ttatacgagc cggaagcata aagtgtaaag cctggggtgc
     7921 ctaatgcggc cgccatagtg actggatatg ttgtgtttta cagtattatg tagtctgttt
     7981 tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt ctcgttcaac
     8041 tttattatac atagttgata attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     8101 ccctggcgtt acccaactta a
//
