LOCUS       sequence-213801-        3639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockout cassette G418.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3639)
  AUTHORS   Paschke P, Knecht DA, Silale A, Traynor D, Williams TD, Thomason PA,
            Insall RH, Chubb JR, Kay RR, Veltman DM
  TITLE     Rapid and efficient genetic engineering of both wild type and axenic
            strains of Dictyostelium discoideum.
  JOURNAL   PLoS One. 2018 May 30;13(5):e0196809. doi: 
            10.1371/journal.pone.0196809. eCollection 2018.
  PUBMED    29847546
REFERENCE   2  (bases 1 to 3639)
  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: "PLoS One.";
            date: "2018-05-30"; volume: "13(5):e0196809. doi"; pages: " 
            10.1371/journal.pone.0196809. eCollection 2018"
COMMENT     Sequence Label: pDM1082
FEATURES             Location/Qualifiers
     source          1..3639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(51..84)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             523..1317
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(577..596)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1187..1206
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(1590..1623)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1709..1813
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1814..1882,1883..2674)
                     /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(2032..2051)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2845..3433
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3334..3353
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3587..3604
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gccggccttc agtaggcaga gctatcaagc ttgatatctt aattaattaa ataacttcgt
       61 atagcataca ttatacgaag ttatagtagt ggatcccaaa attttgtatt ttgtgatgtt
      121 agtgtgtgtc ataattggaa tattgaaaaa aaaaaataaa aaataaaaaa aaaaaaaaaa
      181 agaattaatt aatgattaat catatgacta acattatatt ttcaattttt tttttttttt
      241 ttttccaacc acccaaaagt gatccatttt tttttttttt ttaaataaaa tttgggtcac
      301 ccacakttta atttttttta wttttttttt tttttttttt tttttttaat tttttttttt
      361 tttttcaaaa tttttttttt ttcatataaa aataaatttt ccaattcatt tttttttttt
      421 ttattcaact gtatattatt ataactcttt atttgtattt taaactaatt tcacagctag
      481 caaaaaatgg atggtgaaga tgttcaagct ggatcgtttc gcatgattga acaagatgga
      541 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
      601 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
      661 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
      721 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
      781 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
      841 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
      901 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
      961 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     1021 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     1081 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     1141 atcgactgtg gacggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     1201 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     1261 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     1321 gccgcatcaa tttgatttct tcttaattca atgctaaaaa aaaaaatatt ataaaaaaaa
     1381 aacacacaca acaaataaat taataaatac catttattaa aaaaaaaaaa aaaaaaaaaa
     1441 cctattttaa caattatttt attattttta tttttagttt tgttaaaaaa aaaattatta
     1501 tctttattta taattataaa aaatttatcc aatgatcatt ttttaacttt tttattattt
     1561 agtatattaa ataaaatctc gagagtagta taacttcgta tagcatacat tatacgaagt
     1621 tatgatatca gatctctact ggagtatcca agctgactag ttgatcccgc tacagggcgc
     1681 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1741 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1801 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     1861 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     1921 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     1981 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     2041 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     2101 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     2161 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     2221 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     2281 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     2341 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     2401 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     2461 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     2521 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     2581 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     2641 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     2701 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     2761 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2821 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2881 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2941 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     3001 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3061 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3121 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3181 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3241 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3301 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3361 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3421 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3481 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3541 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3601 cgaggaagcg gaagagcgcc caatacgcaa accgcctct
//
