LOCUS       sequence-215692-        6112 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  safe haven knock-in plasmid for the act5 locus, N-terminal mCherry 
            tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6112)
  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 6112)
  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: pDM1514
FEATURES             Location/Qualifiers
     source          1..6112
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1495..1528
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1802..2839)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
                     MGEAN"
     protein_bind    3275..3308
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4170..4187)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4341..4929)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4421..4440)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5100..5891,5892..5960))
                     /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     5723..5742
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5961..6065)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 actagcgccg gcattacatg tccatttatt agaaggattc aatcaaaaat taacaccagg
       61 tatattacca gatggcatta ataatttata ttctttaata ttggaaaaag aaaaaaataa
      121 ataaacaatc aatcagaaaa gataattgaa aaaaaaaaaa aagaaaatgt ttaataagaa
      181 ttaatttgga agtaaaataa tttttggatg agtaaatatt aataaactca caattaaaat
      241 ttagaatcca aaaaaatgaa aaaaaaaaaa atttttaaaa cagaatggtt gcattcattt
      301 tttttttttt ttttatatct tttgggtatt tttgggtgtt ctatttatct ggataaaaaa
      361 taaaatggaa tatagtttta tatatatatt tttttcgtat caagcttgaa tgtttttatc
      421 aataatatga ttgttggttg ctttatttta tttgaaaaaa aaattaattt ttaaaatatc
      481 ttttttttca ttttatttga aaaaaaaaaa aaaaaaaaat ggttttgaaa atttttaaaa
      541 aaatggttgg gtcacccaca attcttataa aaaaaagcca tttggatagt tttttttttt
      601 ttttttttta ttttttttat tttttttttt ttgagatttg gtataaatac aaaaaaaaaa
      661 ttgtttttaa atcattatta ataaaaactt aaaataaatt atatataaaa aaagatccaa
      721 aaaatggttt caaaaggtga agaagataat atggcaatta ttaaagaatt tatgagattt
      781 aaagttcata tggaaggttc agttaatggt catgaatttg aaattgaagg tgaaggtgaa
      841 ggtagaccat atgaaggtac acaaacagca aaattaaaag ttacaaaagg tggtccatta
      901 ccatttgcat gggatatttt atcaccacaa tttatgtatg gttcaaaagc atatgttaaa
      961 catccagcag atattccaga ttatttaaaa ttatcatttc cagaaggttt taaatgggaa
     1021 agagttatga attttgaaga tggtggtgtt gttacagtta cacaagattc atcattacaa
     1081 gatggtgaat ttatttataa agttaaatta agaggtacaa attttccatc agatggtcca
     1141 gttatgcaaa agaaaacaat gggttgggaa gcatcatcag aaagaatgta tccagaagat
     1201 ggtgcattaa aaggtgaaat taaacaaaga ttaaaattaa aagatggtgg tcattatgat
     1261 gcagaagtta aaacaacata taaagcaaaa aaaccagttc aattaccagg tgcatataat
     1321 gttaatatta aattagatat tacatcacat aatgaagatt atacaattgt tgaacaatat
     1381 gaaagagcag aaggtagaca ttcaacaggt ggtatggatg aattatataa aggtggttca
     1441 ggaggtagta gatcttcaac tagtataaaa taaataaatt gtggatctga tatcataact
     1501 tcgtataatg tatgctatac gaagttatac tactctcgag attttattta atatactaaa
     1561 taataaaaaa gttaaaaaat gatcattgga taaatttttt ataattataa ataaagataa
     1621 taattttttt tttaacaaaa ctaaaaataa aaataataaa ataattgtta aaataggttt
     1681 tttttttttt tttttttttt aataaatggt atttattaat ttatttgttg tgtgtgtttt
     1741 ttttttataa tatttttttt tttagcattg aattaagaag aaatcaaatt gatgcggccg
     1801 cttagttagc ctcccccatc tcccgatccg gacgagtgct ggggcgtcgg tttccactat
     1861 cggcgagtac ttctacacag ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg
     1921 tacgcccgac agtcccggct ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag
     1981 ctgcatcatc gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga
     2041 gcatatacgc ccggagccgc ggcgatcctg caagctccgg atgcctccgc tcgaagtagc
     2101 gcgtctgctg ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt
     2161 attgggaatc cccgaacatc gcctcgctcc agtcaatgac cgctgttatg cggccattgt
     2221 ccgtcaggac attgttggag ccgaaatccg cgtgcacgag gtgccggact tcggggcagt
     2281 cctcggccca aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt
     2341 ccatcacagt ttgccagtga tacacatggg gatcagcaat cgcgcatatg aaatcacgcc
     2401 atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa
     2461 gatcggccgc agcgatcgca tccatggcct ccgcgaccgg ctgcagaaca gcgggcagtt
     2521 cggtttcagg caggtcttgc aacgtgacac cctgtgcacg gcgggagatg caataggtca
     2581 ggctctcgct gaactcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa
     2641 agtgccgata aacataacga tctttgtaga aaccatcggc gcagctattt acccgcagga
     2701 catatccacg ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct
     2761 gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg
     2821 tgagttcagg ctttttcatt aggtcttgtt gagaaatgtt aaattgatcc attttttgct
     2881 agctgtgaaa ttagtttaaa atacaaataa agagttataa taatatacag ttgaataaaa
     2941 aaaaaaaaaa traattggaa aatttatttt tatatraaaa aaaaaaaatt ttraaaaaaa
     3001 aaaaaaaaaw waaaaaaaaa aaaaaaaaaa aaaaaaawwa aaaawaattc aamtgtgggt
     3061 gacccaaatt ttatttaaaa aaaaaaaaaa aatggatcac ttttgggtgg ttggaaaaaa
     3121 aaaaaaaaaa aaattgaaaa tataatgtta gtcatatgat taatcattaa ttaattcttt
     3181 tttttttttt ttttattttt tatttttttt tttcaatatt ccaattatga cacacactaa
     3241 catcacaaaa tacaaaattt tgggatccac tactataact tcgtataatg tatgctatac
     3301 gaagttattt aattaattaa gatatcaagc ttgtccacag aaaatgtttc taaacaaaaa
     3361 aaaaaaaccg agtgatgaaa gcgcttctca caaaattatt tatgtaaaat atttaataaa
     3421 atacattatt taaatctttt ttattttttt tagttgttga tctttatccg actttaaaaa
     3481 taaaaaaatt gtaaaaaaaa aaaaagttta tttgtttaat tttattgttg tttttaattt
     3541 tttacaacca ttaatttata aaaccaacac ctaactataa tgaaatacaa tttgtatctt
     3601 ggaaaaaaaa aaattaataa tataaataaa atgtaaacaa gtatccggtg gaaggaacta
     3661 acagttgcct tatccagttc aaaaaaaaaa aaaaaaaaag aaattaaatt accattttat
     3721 atttaactaa aataaattaa aaggtttatt ttaataatag atttattatt ttttaaaaat
     3781 caataattaa tgttggataa gatgaacgaa taaaagatag agtttcaggt cttttaaatg
     3841 atgaggattt atgaattctt aaaagttttg ggcaagagaa aatgatttta acgattgaac
     3901 catcactaat tgcaaaatct cttgaactaa gagtttttaa gtttgaagca agtggagaat
     3961 tacaaagagc aatcatagag ttatcagtta aatcataaca acaagataaa gataaagaac
     4021 taatatgatg taaattttca atcatacctc taaaagaaac atcattcata ctttttggat
     4081 aaattaaatc taatgaatct aaatttggaa atagtaaacc aatatttggc cggcagaggc
     4141 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4201 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4261 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4321 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4381 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4441 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4501 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4561 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4621 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4681 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4741 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4801 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4861 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4921 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4981 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5041 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5101 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5161 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5221 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5281 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5341 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5401 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5461 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5521 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5581 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5641 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5701 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5761 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5821 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5881 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5941 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6001 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6061 ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcgggat ca
//
