LOCUS       sequence-205380-        6118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  safe haven knock-in plasmid for the act5 locus, C-terminal GFP 
            tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6118)
  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 6118)
  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: pDM1515
FEATURES             Location/Qualifiers
     source          1..6118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1498..1531
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1807..2844)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
                     MGEAN"
     protein_bind    3281..3314
                     /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(4176..4193)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4347..4935)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4427..4446)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5106..5897,5898..5966))
                     /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     5729..5748
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5967..6071)
                     /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 ttttttatat cttttgggta tttttgggtg ttctatttat ctggataaaa
      361 aataaaatgg aatatagttt tatatatata tttttttcgt atcaagcttg aatgttttta
      421 tcaataatat gattgttggt tgctttattt tatttgaaaa aaaaattaat ttttaaaata
      481 tctttttttt cattttattt gaaaaaaaaa aaaaaaaaaa atggttttga aaatttttaa
      541 aaaaatggtt gggtcaccca caattcttat aaaaaaaagc catttggata gttttttttt
      601 tttttttttt tatttttttt attttttttt ttttgagatt tggtataaat acaaaaaaaa
      661 aattgttttt aaatcattat taataaaaac ttaaaataaa ttatatataa aaaaagatct
      721 tcaactagtg gtggttcagg aggtagttca aaaggtgaag aattatttac aggtgttgtt
      781 ccaattttag ttgaattaga tggtgatgtt aatggtcata aattttcagt ttcaggtgaa
      841 ggtgaaggtg atgcaacata tggtaaatta acattaaaat ttatttgtac aacaggtaaa
      901 ttaccagttc catggccaac attagttaca acatttacat atggtgttca atgtttttca
      961 agatatccag atcatatgaa acaacatgat ttttttaaat cagcaatgcc agaaggttat
     1021 gttcaagaaa gaacaatttt ttttaaagat gatggtaatt ataaaacaag agcagaagtt
     1081 aaatttgaag gtgatacatt agttaataga attgaattaa aaggtattga ttttaaagaa
     1141 gatggtaata ttttaggtca taaattagaa tataattata attcacataa tgtttatatt
     1201 atggcagata aacaaaaaaa tggtattaaa gttaatttta aaattagaca taatattgaa
     1261 gatggttcag ttcaattagc agatcattat caacaaaata caccaattgg tgatggtcca
     1321 gttttattac cagataatca ttatttatca acacaatcag cattatcaaa agatccaaat
     1381 gaaaaaagag atcatatggt tttattagaa tttgttacag cagcaggtat tacacatggt
     1441 atggatgaat tatataaata agctagtata aaataaataa attgtggatc tgatatcata
     1501 acttcgtata atgtatgcta tacgaagtta tactactctc gagattttat ttaatatact
     1561 aaataataaa aaagttaaaa aatgatcatt ggataaattt tttataatta taaataaaga
     1621 taataatttt ttttttaaca aaactaaaaa taaaaataat aaaataattg ttaaaatagg
     1681 tttttttttt tttttttttt ttttaataaa tggtatttat taatttattt gttgtgtgtg
     1741 tttttttttt tataatattt ttttttttag cattgaatta agaagaaatc aaattgatgc
     1801 ggccgcttag ttagcctccc ccatctcccg atccggacga gtgctggggc gtcggtttcc
     1861 actatcggcg agtacttcta cacagccatc ggtccagacg gccgcgcttc tgcgggcgat
     1921 ttgtgtacgc ccgacagtcc cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc
     1981 ccaagctgca tcatcgaaat tgccgtcaac caagctctga tagagttggt caagaccaat
     2041 gcggagcata tacgcccgga gccgcggcga tcctgcaagc tccggatgcc tccgctcgaa
     2101 gtagcgcgtc tgctgctcca tacaagccaa ccacggcctc cagaagaaga tgttggcgac
     2161 ctcgtattgg gaatccccga acatcgcctc gctccagtca atgaccgctg ttatgcggcc
     2221 attgtccgtc aggacattgt tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg
     2281 gcagtcctcg gcccaaagca tcagctcatc gagagcctgc gcgacggacg cactgacggt
     2341 gtcgtccatc acagtttgcc agtgatacac atggggatca gcaatcgcgc atatgaaatc
     2401 acgccatgta gtgtattgac cgattccttg cggtccgaat gggccgaacc cgctcgtctg
     2461 gctaagatcg gccgcagcga tcgcatccat ggcctccgcg accggctgca gaacagcggg
     2521 cagttcggtt tcaggcaggt cttgcaacgt gacaccctgt gcacggcggg agatgcaata
     2581 ggtcaggctc tcgctgaact ccccaatgtc aagcacttcc ggaatcggga gcgcggccga
     2641 tgcaaagtgc cgataaacat aacgatcttt gtagaaacca tcggcgcagc tatttacccg
     2701 caggacatat ccacgccctc ctacatcgaa gctgaaagca cgagattctt cgccctccga
     2761 gagctgcatc aggtcggaga cgctgtcgaa cttttcgatc agaaacttct cgacagacgt
     2821 cgcggtgagt tcaggctttt tcattaggtc ttgttgagaa atgttaaatt gatccatttt
     2881 ttgctagctg tgaaattagt ttaaaataca aataaagagt tataataata tacagttgaa
     2941 taaaaaaaaa aaaaatgaat tggaaaattt atttttatat gaagaaaaaa aaattttgaa
     3001 aaaaaaaaaa aaaattaaaa aaaaaaaaaa aaaaaaaaaa aattaaaaat aattcaactg
     3061 tgggtgaccc aaattttatt taaaaaaaaa aaaaaaatgg atcacttttg ggtggttgga
     3121 aaaaaaaaaa aaaaaaaatt gaaaatataa tgttagtcat atgattaatc attaattaat
     3181 tctttttttt ttttttttta ttttttattt tttttttttc aatattccaa ttatgacaca
     3241 cactaacatc acaaaataca aaattttggg atccactact ataacttcgt ataatgtatg
     3301 ctatacgaag ttatttaatt aattaagata tcaagcttgt ccacagaaaa tgtttctaaa
     3361 caaaaaaaaa aaaccgagtg atgaaagcgc ttctcacaaa attatttatg taaaatattt
     3421 aataaaatac attatttaaa tcttttttat tttttttagt tgttgatctt tatccgactt
     3481 taaaaataaa aaaattgtaa aaaaaaaaaa agtttatttg tttaatttta ttgttgtttt
     3541 taatttttta caaccattaa tttataaaac caacacctaa ctataatgaa atacaatttg
     3601 tatcttggaa aaaaaaaaat taataatata aataaaatgt aaacaagtat ccggtggaag
     3661 gaactaacag ttgccttatc cagttcaaaa aaaaaaaaaa aaaaagaaat taaattacca
     3721 ttttatattt aactaaaata aattaaaagg tttattttaa taatagattt attatttttt
     3781 aaaaatcaat aattaatgtt ggataagatg aacgaataaa agatagagtt tcaggtcttt
     3841 taaatgatga ggatttatga attcttaaaa gttttgggca agagaaaatg attttaacga
     3901 ttgaaccatc actaattgca aaatctcttg aactaagagt ttttaagttt gaagcaagtg
     3961 gagaattaca aagagcaatc atagagttat cagttaaatc ataacaacaa gataaagata
     4021 aagaactaat atgatgtaaa ttttcaatca tacctctaaa agaaacatca ttcatacttt
     4081 ttggataaat taaatctaat gaatctaaat ttggaaatag taaaccaata tttggccggc
     4141 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4201 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4261 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4321 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4381 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4441 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4501 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4561 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4621 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4681 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4741 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     4801 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4861 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4921 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4981 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5041 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5101 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5161 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5221 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5281 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5341 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5401 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5461 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5521 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5581 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5641 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5701 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5761 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5821 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5881 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5941 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6001 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6061 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag cgggatca
//
