LOCUS       sequence-12386-e        7401 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 7401)
  AUTHORS   Semple JI, Garcia-Verdugo R, Lehner B
  TITLE     Rapid selection of transgenic C. elegans using antibiotic 
            resistance.
  JOURNAL   Nat Methods. 2010 Sep;7(9):725-7. doi: 10.1038/nmeth.1495. Epub 2010
            Aug 22.
  PUBMED    20729840
REFERENCE   2  (bases 1 to 7401)
  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.1495"; journalName: "Nat Methods"; date: "2010-09"; 
            volume: "7"; issue: "9"; pages: "725-7"
COMMENT     Sequence Label: pBCN22-R4R3
FEATURES             Location/Qualifiers
     source          1..7401
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(530..1240)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(808..827)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1077..1095
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2305..2324)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2903..2923
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3585..3607
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3645..3663)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3731..3835
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3836..3904,3905..4696)
                     /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(4054..4073)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4867..5455
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5356..5375
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5581..5603
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5600..5616
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5600..5616
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5636..5759
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(5800..6105)
                     /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
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(5902..5921)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(6425..7105)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     7020..7039
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(7159..7165,7166..7183,7184..7189))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(7214..7337)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7345..7362)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7345..7361)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7354..7376)
                     /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 gccctgtgtg cggttttttc
      181 tatgatgttt caaaaaaatt tatcagaagt aaaaaacata agaagtgtac acagaacatt
      241 gtgtagattt tgggatagaa ttattattaa tggggggagg gagcacaatt tttttcgact
      301 ttttattacg aattaattct atttttgaat ctacacaatt tcattgttag aggtgactta
      361 aaagaagcta aaaaacaaag aaattaagag aagttttttg ataataacaa aaataggggg
      421 tgggagcaca gggagaaaga gcatgtaggg atgttgaaga gtaattggac ttagaagtca
      481 gaggcacggg cgcgagatgg cgatctgatg acagcggccg atgcggagct tacttgtaca
      541 gctcgtccat gccgccggtg gagtggcggc cctcggcgcg ttcgtactgt tccacgatgg
      601 tgtagtcctc gttgtgggag gtgatgtcca acttgatgtt gacgttgtag gcgccgggca
      661 gctgcacggg cttcttggcc ttgtaggtgg tcttgacctc agcgtcgtag tggccgccgt
      721 ccttcagctt cagcctctgc ttgatctcgc ccttcagggc gccgtcctcg gggtacatcc
      781 gctcggagga ggcctcccag cccatggtct tcttctgcat tacggggccg tcggagggga
      841 agttggtgcc gcgcagcttc accttgtaga tgaactcgcc gtcctgcagg gaggagtcct
      901 gggtcacggt caccacgccg ccgtcctcga agttcatcac gcgctcccac ttgaagccct
      961 cggggaagga cagcttcaag tagtcgggga tgtcggcggg gtgcttcacg taggccttgg
     1021 agccgtacat gaactgaggg gacaggatgt cccaggcgaa gggcaggggg ccacccttgg
     1081 tcaccttcag cttggcggtc tgggtgccct cgtaggggcg gccctcgccc tcgccctcga
     1141 tctcgaactc gtggccgttc acggagccct ccatgtgcac cttgaagcgc atgaactcct
     1201 tgatgatggc catgttatcc tcctcgccct tgctcaccat ggtcgagggt taaaatgaaa
     1261 agtggtggag agaagtcaac cgctcggtgg gatgcgcgag cctggagata actttgattt
     1321 tcggcgagcc cttctttttc cctgggtggg tggattggga ggcggggata tccaattgta
     1381 attctaggta gctgaaaagt gtctagagtc gaccaagtct cgttgtttgc cgtcggatgt
     1441 ctgcccaagt ctcgttgttt gccgtcggat gtctgcccaa gtctcgttgt ttgccgtcgg
     1501 atgtctgccc aagtctcgtt gtttgccgtc ggatgtctgc ccaagtctcg ttgtttgccg
     1561 tcggatgtct gcccaagtct cgttgtttgc cgtcggatgt ctgcccaagt ctcgttgttt
     1621 gccgtcggat gtctgcccaa ggcctgcagg aattcgatat caagcttggt accgagctcg
     1681 ggggcccccc tcgaggggag aaaatgcgga gagaagaacc cagcatctta ataaaattat
     1741 ataattttga ataaaatcaa attaaaaata agattaattg aaacacaaca atagaatgca
     1801 gaaaattact cataagaaat aatttatttt gaaagaagtt gtgtcgattt acgataaaaa
     1861 ctacacgcaa acgaggctcc gcgtttacac cgtttggcaa tggagcgcgc ttgctattag
     1921 attttaggcc agctcaacga aagtttaatt tatttttctt ctttttttcc atttttttga
     1981 gtaaaatatc gaattttttg tattaattct gtaaatttta taatttgcaa ctattttggc
     2041 gtattctgct tgtttttatc agcgaatttt tatgcatttc agtcaaaagt ttgagcagga
     2101 aaaatgacat ttaggacttt gtaaaattgt attcttaaat ttgcttgaaa acctacgtgt
     2161 acaattgatt ttattgaatt tttcttacgt ttcccgttgt atcttattgt atttaacttt
     2221 catcataaaa taatttattt ccaggtaaaa tatccgacgc tctcgtggat ccccgggatt
     2281 ggctcagaaa aaaaggtacc gaccatgacc gagtacaagc ccacggtgcg cctcgccacc
     2341 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     2401 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     2461 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcggtaagtt taaacatata
     2521 tatactaact aaccctgatt atttaaattt tcagcggacg acggcgccgc ggtggcggtc
     2581 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     2641 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     2701 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgacgtaagt
     2761 ttaaacatga ttttactaac taactaatct gatttaaatt ttcagcacca gggcaagggt
     2821 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     2881 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     2941 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     3001 gcctgatgag ctagccggcc atacaagtaa tccggatgat cgacgccaac gtcgttgaat
     3061 tttcaaattt taaatactga atatttgttt tttttcctat tatttattta ttctctttgt
     3121 gttttttttc ttgctttcta aaaaattaat tcaatccaaa tctaaacatt tttttttctc
     3181 tttccgtctc ccaattcgta ttccgctcct ctcatctgaa cacaatgtgc aagtttattt
     3241 atcttctcgc tttcatttca ttaggacgtg gggggaattg gtggaagggg gaaacacaca
     3301 aaaggatgat ggaaatgaaa taaggacaca caatatgcaa caacattcaa ttcagaaata
     3361 tggaggaagg tttaaaagaa aacataaaaa tatatagagg aggaaggaaa actagttcta
     3421 gagcggccgc caccgcggtg gagctcccat atggtgcact ctcagtgcaa tctgctctga
     3481 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     3541 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3601 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     3661 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     3721 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3781 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3841 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3901 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3961 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4021 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     4081 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4141 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4201 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4261 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4321 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     4381 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4441 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4501 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4561 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4621 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4681 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4741 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4801 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4861 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4921 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4981 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     5041 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5101 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5161 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5221 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5281 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5341 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5401 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5461 cagcaacgcg gcctttttac ggttcctggt cttttgctgg cctttgctca catgttcttt
     5521 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     5581 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctatcaact
     5641 ttgtatagaa aagttgaacg agaaacgtaa aatgatataa atatcaatat attaaattag
     5701 attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca gtcactatgg
     5761 tcgacctgca gactggctgt gtataaggga gcctgacatt tatattcccc agaacatcag
     5821 gttaatggcg tttttgatgt cattttcgcg gtggctgaga tcagccactt cttccccgat
     5881 aacggagacc ggcacactgg ccatatcggt ggtcatcatg cgccagcttt catccccgat
     5941 atgcaccacc gggtaaagtt cacgggggac tttatctgac agcagacgtg cactggccag
     6001 ggggatcacc atccgtcgcc cgggcgtgtc aataatatca ctctgtacat ccacaaacag
     6061 acgataacgg ctctctcttt tataggtgta aaccttaaac tgcatttcac cagcccctgt
     6121 tctcgtcggc aaaagagccg ttcatttcaa taaaccgggc gacctcagcc atcccttcct
     6181 gattttccgc tttccagcgt tcggcacgca gacgacgggc ttcattctgc atggttgtgc
     6241 ttaccgaacc ggagatattg acatcatata tgccttgagc aactgatagc tgtcgctgtc
     6301 aactgtcact gtaatacgct gcttcatagc atacctcttt ttgacatact tcgggtatac
     6361 atatcagtat atattcttat accgcaaaaa tcagcgcgca aatacgcata ctgttatctg
     6421 gcttttagta agccggatcc tctagattac gccccgcctg ccactcatcg cagtactgtt
     6481 gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa
     6541 tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg
     6601 gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg
     6661 gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt
     6721 caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt
     6781 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt
     6841 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag
     6901 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct
     6961 ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag
     7021 caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg
     7081 tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat ctcgacggat
     7141 cctaactcaa aatccacaca ttatacgagc cggaagcata aagtgtaaag cctggggtgc
     7201 ctaatgcggc cgccatagtg actggatatg ttgtgtttta cagtattatg tagtctgttt
     7261 tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt ctcgttcaac
     7321 tttattatac atagttgata attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     7381 ccctggcgtt acccaactta a
//
