LOCUS       sequence-335009-        2415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Alpha level R6K_ origin GB Cloning vector, Conditionally replicative
            ori, 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 2415)
  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 2415)
  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-Alpha1
FEATURES             Location/Qualifiers
     source          1..2415
                     /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        274..321
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(702..721)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        complement(772..790)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(773..790)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(808..824)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(808..824)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      895..967
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      1023..1379
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(1493..2308)
                     /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(1493..2308)
                     /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     2216..2235
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2309..2411)
                     /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 atagggttcg aattctatcg
      241 gtaagcacaa atcgtctcag gagagagacc cctgttgaca attaatcatc ggcatagtat
      301 atcggcatag tataatacga caaggtgagg aactaaaccc aggaggcaga gatcatgacc
      361 atgattacgc caagctatct gggcgatact atcgaatact ctagcttgca tgcgtgcagg
      421 tcgactctag aagatccccg ggtaccgagc tctaattcgc cgtacagcga gtcttactac
      481 aattctctgg cggtggtctt gcaacgtcgt gactgggaaa accctggcgt tacccaactt
      541 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
      601 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gacgcctgat gcggtatttt
      661 ctccttacgt tactgtgcgg tatttcacac gtgcacctca gtacaatctg ctctgatgcc
      721 gcatagggtc tctcgctgtc atgagacgaa ttctgacttg tattccgcgg ttctatagtg
      781 tcacctaaat agcttggcgt aatttaggtc atagctgttt cctgtcggac caaaacgaaa
      841 aaaggccccc ctttcgggag gcctcttttc tggaatttgg taccgagcag gcatcaaata
      901 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggttgaa
      961 cgctctctga cgtaccgcgg ccgcgtagag gatctgttga tcagcagttc aacctgttga
     1021 tagtacgtac taagctctca tgtttcacgt actaagctct catgtttaac gtactaagct
     1081 ctcatgttta acgaactaaa ccctcatggc taacgtacta agctctcatg gctaacgtac
     1141 taagctctca tgtttcacgt actaagctct catgtttgaa caataaaatt aatataaatc
     1201 agcaacttaa atagcctcta aggttttaag ttttataaga aaaaaaagaa tatataaggc
     1261 ttttaaagct tttaaggttt aacggttgtg gacaacaagc cagggatgta acgcactgag
     1321 aagcccttag agcctctcaa agcaattttg agtgacacag gaacacttaa cggctgacat
     1381 gtgatcatgt ttgacagctt atcatcgaat ttctgccatt catccgctta ttatcactta
     1441 ttcaggcgta gcaaccaggc gtttaagggc accaataact gccttaaaaa aattagaaaa
     1501 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     1561 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     1621 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctatcaatt
     1681 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgact actgaatccg
     1741 gtgagaatgg caaaagtttg tgcatttctt tccagacttg ttcaacaggc cagccattac
     1801 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     1861 cgagtcgaaa tacgcggtcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     1921 ggcgcaggaa cactgccagc gcatcaacaa tgttttcacc tgaatcagga tattcttcta
     1981 atacctggaa tgctgttttc cctgggatcg cagtggtgag taaccacgca tcatcaggag
     2041 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     2101 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     2161 gcgcatcggg cttcccatac aatcggtaga ttgtcgcacc tgattgcccg acattatcgc
     2221 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggccttgagc
     2281 aagacgtttc ccgttgaata tggctcattt tagcttcctt agctcctgaa aatctcgata
     2341 actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta
     2401 cgtgccgatc ataac
//
