LOCUS       sequence-335011-        2399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Alpha level R6K_ origin GB Cloning vector, Conditionally replicative
            ori, requires pir gene expression requires pir gene expression 
            requires EC100D or EC100D116 bacterial strain (DH10B derived) uses 
            blue-white screening.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2399)
  AUTHORS   Matinyan N, Karkhanis MS, Gonzalez Y, Jain A, Saltzman A, 
            Malovannaya A, Sarrion-Perdigones A, Dierick HA, Venken KJT
  TITLE     Multiplexed drug-based selection and counterselection genetic 
            manipulations in Drosophila.
  JOURNAL   Cell Rep. 2021 Sep 14;36(11):109700. doi: 
            10.1016/j.celrep.2021.109700.
  PUBMED    34525356
REFERENCE   2  (bases 1 to 2399)
  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"; journalName: "Cell Rep.";
            date: "2021-09-14"; volume: "36(11):109700. doi"; pages: " 
            10.1016/j.celrep.2021.109700"
COMMENT     Sequence Label: pR6Kg-Alpha2
FEATURES             Location/Qualifiers
     source          1..2399
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(15..42)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     183..200
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     184..200
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     207..226
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        207..225
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        261..308
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(689..708)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        complement(756..774)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(757..774)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(792..808)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(792..808)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      879..951
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      1007..1363
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(1477..2292)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1477..2292)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2200..2219
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2293..2395)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 attgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttaatt tgatgcctgg
       61 cagtttatgg cgggcgtcct gcccgccacc ctttcagcca aaaaacttaa gaccgccggt
      121 cttgtccact accttgcagt aatgcggtgg acaggatcgg cggttttctt ttctcttctc
      181 aatgtaaaac gacggccagt gaattgtaat acgactcact atagggttcc agctgcgtct
      241 cagtcaggag agagacccct gttgacaatt aatcatcggc atagtatatc ggcatagtat
      301 aatacgacaa ggtgaggaac taaacccagg aggcagagat catgaccatg attacgccaa
      361 gctatctggg cgatactatc gaatactcta gcttgcatgc gtgcaggtcg actctagaag
      421 atccccgggt accgagctct aattcgccgt acagcgagtc ttactacaat tctctggcgg
      481 tggtcttgca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
      541 cacatccccc tttcgccagt tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
      601 aacagttgcg cagcctgaat ggcgaatgac gcctgatgcg gtattttctc cttacgttac
      661 tgtgcggtat ttcacacgtg cacctcagta caatctgctc tgatgccgca tagggtctct
      721 cgcttgagac gcagctgtga cttgtattcc gcggttctat agtgtcacct aaatagcttg
      781 gcgtaattta ggtcatagct gtttcctgtc ggaccaaaac gaaaaaaggc ccccctttcg
      841 ggaggcctct tttctggaat ttggtaccga gcaggcatca aataaaacga aaggctcagt
      901 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt tgaacgctct ctgacgtacc
      961 gcggccgcgt agaggatctg ttgatcagca gttcaacctg ttgatagtac gtactaagct
     1021 ctcatgtttc acgtactaag ctctcatgtt taacgtacta agctctcatg tttaacgaac
     1081 taaaccctca tggctaacgt actaagctct catggctaac gtactaagct ctcatgtttc
     1141 acgtactaag ctctcatgtt tgaacaataa aattaatata aatcagcaac ttaaatagcc
     1201 tctaaggttt taagttttat aagaaaaaaa agaatatata aggcttttaa agcttttaag
     1261 gtttaacggt tgtggacaac aagccaggga tgtaacgcac tgagaagccc ttagagcctc
     1321 tcaaagcaat tttgagtgac acaggaacac ttaacggctg acatgtgatc atgtttgaca
     1381 gcttatcatc gaatttctgc cattcatccg cttattatca cttattcagg cgtagcaacc
     1441 aggcgtttaa gggcaccaat aactgcctta aaaaaattag aaaaactcat cgagcatcaa
     1501 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     1561 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     1621 gtctgcgatt ccgactcgtc caacatcaat acaacctatc aatttcccct cgtcaaaaat
     1681 aaggttatca agtgagaaat caccatgagt gactactgaa tccggtgaga atggcaaaag
     1741 tttgtgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     1801 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagtc gaaatacgcg
     1861 gtcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     1921 cagcgcatca acaatgtttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     1981 tttccctggg atcgcagtgg tgagtaacca cgcatcatca ggagtacgga taaaatgctt
     2041 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     2101 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     2161 atacaatcgg tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     2221 atataaatca gcatccatgt tggaatttaa tcgcggcctt gagcaagacg tttcccgttg
     2281 aatatggctc attttagctt ccttagctcc tgaaaatctc gataactcaa aaaatacgcc
     2341 cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcataac
//
