LOCUS       sequence-275144-        5825 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides negative selection cassette on the vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5825)
  TITLE     Minoru Ko Lab plasmids
REFERENCE   2  (bases 1 to 5825)
  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"
COMMENT     Sequence Label: pDEST-R4R3-MC1DTA
FEATURES             Location/Qualifiers
     source          1..5825
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    37..160
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(508..813)
                     /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(610..629)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1133..1816)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     1731..1750
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1817..1919)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2065..2188)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2196..2213)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2196..2212)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2205..2227)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2663..3235
                     /codon_start=1
                     /gene="Corynebacterium diphtheriae tox"
                     /product="diphtheria toxin fragment A"
                     /label=DTA
                     /translation="DDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYD
                     DDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKEL
                     GLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEI
                     NFETRGKRGQDAMYEYMAQACAGNRVRR"
     intron          3312..3377
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     primer_bind     3810..3832
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3870..3888)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3956..4060
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4061..4129,4130..4921)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGSDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4279..4298)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5092..5680
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5581..5600
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(5807..5825,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca agctatcaac tttgtataga aaagttgaac
       61 gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga
      121 ctacataata ctgtaaaaca caacatatcc agtcactatg gtcgaccgac agccttccaa
      181 atgttcttct caaacggaat cgtcgtatcc agcctactcg ctattgtcct caatgccgta
      241 ttaaatcata aaaagaaata agaaaaagag gtgcgagcct cttttttgtg tgacaaaata
      301 aaaacatcta cctattcata tacgctagtg tcatagtcct gaaaatcatc tgcatcaaga
      361 acaatttcac aactcttata cttttctctt acaagtcgtt cggcttcatc tggattttca
      421 gcctctatac ttactaaacg tgataaagtt tctgtaattt ctactgtatc gacctgcaga
      481 ctggctgtgt ataagggagc ctgacattta tattccccag aacatcaggt taatggcgtt
      541 tttgatgtca ttttcgcggt ggctgagatc agccacttct tccccgataa cggagaccgg
      601 cacactggcc atatcggtgg tcatcatgcg ccagctttca tccccgatat gcaccaccgg
      661 gtaaagttca cgggagactt tatctgacag cagacgtgca ctggccaggg ggatcaccat
      721 ccgtcgcccg ggcgtgtcaa taatatcact ctgtacatcc acaaacagac gataacggct
      781 ctctctttta taggtgtaaa ccttaaactg catttcacca gcccctgttc tcgtcagcaa
      841 aagagccgtt catttcaata aaccgggcga cctcagccat cccttcctga ttttccgctt
      901 tccagcgttc ggcacgcaga cgacgggctt cattctgcat ggttgtgctt accagaccgg
      961 agatattgac atcatatatg ccttgagcaa ctgatagctg tcgctgtcaa ctgtcactgt
     1021 aatacgctgc ttcatagcat acctcttttt gacatacttc gggtatacat atcagtatat
     1081 attcttatac cgcaaaaatc agcgcgcaaa tacgcatact gttatctggc ttttagtaag
     1141 ccggatccac gcggcgttta cgccccccct gccactcatc gcagtactgt tgtaattcat
     1201 taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg
     1261 gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga
     1321 agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg
     1381 agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac
     1441 acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc
     1501 agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat
     1561 cccatatcac cagctcaccg tctttcattg ccatacggaa ttccggatga gcattcatca
     1621 ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct
     1681 ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact
     1741 gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag
     1801 tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata
     1861 cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa
     1921 cgtctcattt tcgccaaaag ttggcccagg gcttcccggt atcaacaggg acaccaggat
     1981 ttatttattc tgcgaagtga tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg
     2041 gtgatgctgc caacttacgg ccgccatagt gactggatat gttgtgtttt acagtattat
     2101 gtagtctgtt ttttatgcaa aatctaattt aatatattga tatttatatc attttacgtt
     2161 tctcgttcaa ctttattata catagttgat aattcactgg ccgtcgtttt acaacgtcgt
     2221 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2281 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2341 aatggcgaat ggcgccgctt atcgataccg tcgagcagtg tggttttcaa gaggaagcaa
     2401 aaagcctctc cacccaggcc tggaatgttt ccacccaatg tcgagcagtg tgattttgca
     2461 agaggaagca aaaagcctct ccacccaggc ctggaatgtt tccacccaat gtcgagcaaa
     2521 ccccgcccag cgtcttgtca ttggcgaatt cgaacacgca gatgcagtcg gggcggcgcg
     2581 gtcccaggtc cacttcgcat attaaggtga cgcgtgtggc ctcgaacacc gagcgaccct
     2641 gcaggtcctc gccatggatc ctgatgatgt tgttgattct tctaaatctt ttgtgatgga
     2701 aaacttttct tcgtaccacg ggactaaacc tggttatgta gattccattc aaaaaggtat
     2761 acaaaagcca aaatctggta cacaaggaaa ttatgacgat gattggaaag ggttttatag
     2821 taccgacaat aaatacgacg ctgcgggata ctctgtagat aatgaaaacc cgctctctgg
     2881 aaaagctgga ggcgtggtca aagtgacgta tccaggactg acgaaggttc tcgcactaaa
     2941 agtggataat gccgaaacta ttaagaaaga gttaggttta agtctcactg aaccgttgat
     3001 ggagcaagtc ggaacggaag agtttatcaa aaggttcggt gatggtgctt cgcgtgtagt
     3061 gctcagcctt cccttcgctg aggggagttc tagcgttgaa tatattaata actgggaaca
     3121 ggcgaaagcg ttaagcgtag aacttgagat taattttgaa acccgtggaa aacgtggcca
     3181 agatgcgatg tatgagtata tggctcaagc ctgtgcagga aatcgtgtca ggcgatctct
     3241 ttgtgaagga accttacttc tgtggtgtga cataattgga caaactacct acagagattt
     3301 aaagctctaa ggtaaatata aaatttttaa gtgtataatg tgttaaacta ctgattctaa
     3361 ttgtttgtgt attttagatt ccaacctatg gaactgatga atgggagcag tggtggaatg
     3421 cagatccact agagtcgagg gatgcagaaa ttgatgatct attaaacaat aaagatgtcc
     3481 actaaaatgg aagtttttcc tgtcatactt tgttaagaag ggtgagaaca gagtacctac
     3541 attttgaatg gaaggattgg agctacgggg gtgggggtgg ggtgggatta gataaatgcc
     3601 tgctctttac tgaaggctct ttactattgc tttatgataa tgtttcatag ttggatatca
     3661 taatttaaac aacatatggt gcactctcag tgcaatctgc tctgatgccg catagttaag
     3721 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3781 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3841 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     3901 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     3961 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4021 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4081 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     4141 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     4201 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     4261 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     4321 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     4381 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     4441 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     4501 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     4561 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     4621 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     4681 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4741 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     4801 ggggtcagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4861 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4921 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4981 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5041 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5101 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5161 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5221 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5281 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5341 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5401 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5461 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5521 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5581 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5641 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5701 tttacggttc ctggtctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5761 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccagcg gataacaatt
     5821 tcaca
//
