LOCUS       sequence-216008-        6118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  safe haven knock-in plasmid for the act5 locus, N-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: pDM1513
FEATURES             Location/Qualifiers
     source          1..6118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1501..1534
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1808..2845)
                     /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 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 attyttataa aaaaaagcca tttggatagt tttttttttt
      601 ttttttttta ttttttttat tttttttttt ttgagatttg gtataaatac aaaaaaaaaa
      661 ttgtttttaa atcattatta ataaaaactt aaaataaatt atatataaaa aaagatccaa
      721 aaaatgtcaa aaggtgaaga attatttaca ggtgttgttc caattttagt tgaattagat
      781 ggtgatgtta atggtcataa attttcagtt tcaggtgaag gtgaaggtga tgcaacatat
      841 ggtaaattaa cattaaaatt tatttgtaca acaggtaaat taccagttcc atggccaaca
      901 ttagttacaa catttacata tggtgttcaa tgtttttcaa gatatccaga tcatatgaaa
      961 caacatgatt tttttaaatc agcaatgcca gaaggttatg ttcaagaaag aacaattttt
     1021 tttaaagatg atggtaatta taaaacaaga gcagaagtta aatttgaagg tgatacatta
     1081 gttaatagaa ttgaattaaa aggtattgat tttaaagaag atggtaatat tttaggtcat
     1141 aaattagaat ataattataa ttcacataat gtttatatta tggcagataa acaaaaaaat
     1201 ggtattaaag ttaattttaa aattagacat aatattgaag atggttcagt tcaattagca
     1261 gatcattatc aacaaaatac accaattggt gatggtccag ttttattacc agataatcat
     1321 tatttatcaa cacaatcagc attatcaaaa gatccaaatg aaaaaagaga tcatatggtt
     1381 ttattagaat ttgttacagc agcaggtatt acacatggta tggatgaatt atataaaggt
     1441 ggttcaggag gtagtagatc ttcaactagt ataaaataaa taaattgtgg atctgatatc
     1501 ataacttcgt ataatgtatg ctatacgaag ttatactact ctcgagattt tatttaatat
     1561 actaaataat aaaaaagtta aaaaatgatc attggataaa ttttttataa ttataaataa
     1621 agataataat ttttttttta acaaaactaa aaataaaaat aataaaataa ttgttaaaat
     1681 aggttttttt tttttttttt ttttttaata aatggtattt attaatttat ttgttgtgtg
     1741 tgtttttttt ttataatatt ttttttttta gcattgaatt aagaagaaat caaattgatg
     1801 cggccgctta gttagcctcc cccatctccc gatccggacg agtgctgggg cgtcggtttc
     1861 cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt ctgcgggcga
     1921 tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat cgaccctgcg
     1981 cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg tcaagaccaa
     2041 tgcggagcat atacgcccgg agccgcggcg atcctgcaag ctccggatgc ctccgctcga
     2101 agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag atgttggcga
     2161 cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct gttatgcggc
     2221 cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc cggacttcgg
     2281 ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac gcactgacgg
     2341 tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg catatgaaat
     2401 cacgccatgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac ccgctcgtct
     2461 ggctaagatc ggccgcagcg atcgcatcca tggcctccgc gaccggctgc agaacagcgg
     2521 gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg gagatgcaat
     2581 aggtcaggct ctcgctgaac tccccaatgt caagcacttc cggaatcggg agcgcggccg
     2641 atgcaaagtg ccgataaaca taacgatctt tgtagaaacc atcggcgcag ctatttaccc
     2701 gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct tcgccctccg
     2761 agagctgcat caggtcggag acgctgtcga acttttcgat cagaaacttc tcgacagacg
     2821 tcgcggtgag ttcaggcttt ttcattaggt cttgttgaga aatgttaaat tgatccattt
     2881 tttgctagct gtgaaattag tttaaaatac aaataaagag ttataataat atacagttga
     2941 ataaaaaaaa aaaaaatraa ttggaaaatt tatttttata traaaaaaaa aaaattttra
     3001 aaaaaaaaaa aaaaawwaaa aaaaaaaaaa aaaaaaaaaa aaaawaaaaa aaattmaamt
     3061 gtgggtgacc caaattttat ttaaaaaaaa aaaaaaaatg gatcactttt gggtggttgg
     3121 aaaaaaaaaa aaaaaaaaat tgaaaatata atgttagtca tatgattaat cattaattaa
     3181 ttcttttttt tttttttttt attttttatt 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 ctagaagaac 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
//
