LOCUS       sequence-215719-        6113 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 6113)
  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 6113)
  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..6113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1496..1529
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1803..2840)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
                     MGEAN"
     protein_bind    3276..3309
                     /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(4171..4188)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4342..4930)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4422..4441)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5101..5892,5893..5961))
                     /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     5724..5743
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5962..6066)
                     /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 gtyacccaca attyttataa aaaaaagcca tttggatagt tttttttttt
      601 ttttttttta ttttttttat tttttttttt ttgagatttg gtataaatac aaaaaaaaaa
      661 ttgtttttaa atcattatta ataaaaactt aaaataaatt atatataaaa aaagatcttc
      721 aactagtggt ggttcaggag gtagttcaaa aggtgaagaa ttatttacag gtgttgttcc
      781 aattttagtt gaattagatg gtgatgttaa tggtcataaa ttttcagttt caggtgaagg
      841 tgaaggtgat gcaacatatg gtaaattaac attaaaattt atttgtacaa caggtaaatt
      901 accagttcca tggccaacat tagttacaac atttacatat ggtgttcaat gtttttcaag
      961 atatccagat catatgaaac aacatgattt ttttaaatca gcaatgccag aaggttatgt
     1021 tcaagaaaga acaatttttt ttaaagatga tggtaattat aaaacaagag cagaagttaa
     1081 atttgaaggt gatacattag ttaatagaat tgaattaaaa ggtattgatt ttaaagaaga
     1141 tggtaatatt ttaggtcata aattagaata taattataat tcacataatg tttatattat
     1201 ggcagataaa caaaaaaatg gtattaaagt taattttaaa attagacata atattgaaga
     1261 tggttcagtt caattagcag atcattatca acaaaataca ccaattggtg atggtccagt
     1321 tttattacca gataatcatt atttatcaac acaatcagca ttatcaaaag atccaaatga
     1381 aaaaagagat catatggttt tattagaatt tgttacagca gcaggtatta cacatggtat
     1441 ggatgaatta tataaataag ctagtataaa ataaataaat tgtggatctg atatcataac
     1501 ttcgtataat gtatgctata cgaagttata ctactctcga gattttattt aatatactaa
     1561 ataataaaaa agttaaaaaa tgatcattgg ataaattttt tataattata aataaagata
     1621 ataatttttt ttttaacaaa actaaaaata aaaataataa aataattgtt aaaataggtt
     1681 tttttttttt tttttttttt taataaatgg tatttattaa tttatttgtt gtgtgtgttt
     1741 tttttttata atattttttt ttttagcatt gaattaagaa gaaatcaaat tgatgcggcc
     1801 gcttagttag cctcccccat ctcccgatcc ggacgagtgc tggggcgtcg gtttccacta
     1861 tcggcgagta cttctacaca gccatcggtc cagacggccg cgcttctgcg ggcgatttgt
     1921 gtacgcccga cagtcccggc tccggatcgg acgattgcgt cgcatcgacc ctgcgcccaa
     1981 gctgcatcat cgaaattgcc gtcaaccaag ctctgataga gttggtcaag accaatgcgg
     2041 agcatatacg cccggagccg cggcgatcct gcaagctccg gatgcctccg ctcgaagtag
     2101 cgcgtctgct gctccataca agccaaccac ggcctccaga agaagatgtt ggcgacctcg
     2161 tattgggaat ccccgaacat cgcctcgctc cagtcaatga ccgctgttat gcggccattg
     2221 tccgtcagga cattgttgga gccgaaatcc gcgtgcacga ggtgccggac ttcggggcag
     2281 tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga cggacgcact gacggtgtcg
     2341 tccatcacag tttgccagtg atacacatgg ggatcagcaa tcgcgcatat gaaatcacgc
     2401 catgtagtgt attgaccgat tccttgcggt ccgaatgggc cgaacccgct cgtctggcta
     2461 agatcggccg cagcgatcgc atccatggcc tccgcgaccg gctgcagaac agcgggcagt
     2521 tcggtttcag gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat gcaataggtc
     2581 aggctctcgc tgaactcccc aatgtcaagc acttccggaa tcgggagcgc ggccgatgca
     2641 aagtgccgat aaacataacg atctttgtag aaaccatcgg cgcagctatt tacccgcagg
     2701 acatatccac gccctcctac atcgaagctg aaagcacgag attcttcgcc ctccgagagc
     2761 tgcatcaggt cggagacgct gtcgaacttt tcgatcagaa acttctcgac agacgtcgcg
     2821 gtgagttcag gctttttcat taggtcttgt tgagaaatgt taaattgatc cattttttgc
     2881 tagctgtgaa attagtttaa aatacaaata aagagttata ataatataca gttgaataaa
     2941 aaaaaaaaaa atgaattgga aaatttattt ttatatraaa aaaaaaaaat tttraaaaaa
     3001 aaaaaaaaaa wwaaaaaaaa aaaaaaaaaa aaaaaaaatw aaaaawaatt maamtgtggg
     3061 tgacccaaat tttatttaaa aaaaaaaaaa aaatggatca cttttgggtg gttggaaaaa
     3121 aaaaaaaaaa aaaattgaaa atataatgtt agtcatatga ttaatcatta attaattctt
     3181 tttttttttt tttttatttt ttattttttt ttttcaatat tccaattatg acacacacta
     3241 acatcacaaa atacaaaatt ttgggatcca ctactataac ttcgtataat gtatgctata
     3301 cgaagttatt taattaatta agatatcaag cttgtccaca gaaaatgttt ctaaacaaaa
     3361 aaaaaaaacc gagtgatgaa agcgcttctc acaaaattat ttatgtaaaa tatttaataa
     3421 aatacattat ttaaatcttt tttatttttt ttagttgttg atctttatcc gactttaaaa
     3481 ataaaaaaat tgtaaaaaaa aaaaaagttt atttgtttaa ttttattgtt gtttttaatt
     3541 ttttacaacc attaatttat aaaaccaaca cctaactata atgaaataca atttgtatct
     3601 tggaaaaaaa aaaattaata atataaataa aatgtaaaca agtatccggt ggaaggaact
     3661 aacagttgcc ttatccagtt caaaaaaaaa aaaaaaaaaa gaaattaaat taccatttta
     3721 tatttaacta aaataaatta aaaggtttat tttaataata gatttattat tttttaaaaa
     3781 tcaataatta atgttggata agatgaacga ataaaagata gagtttcagg tcttttaaat
     3841 gatgaggatt tatgaattct taaaagtttt gggcaagaga aaatgatttt aacgattgaa
     3901 ccatcactaa ttgcaaaatc tcttgaacta agagttttta agtttgaagc aagtggagaa
     3961 ttacaaagag caatcataga gttatcagtt aaatcataac aacaagataa agataaagaa
     4021 ctaatatgat gtaaattttc aatcatacct ctaaaagaaa catcattcat actttttgga
     4081 taaattaaat ctaatgaatc taaatttgga aatagtaaac caatatttgg ccggcagagg
     4141 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4201 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4261 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4321 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4381 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4441 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4501 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4561 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4621 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4681 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4741 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     4801 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4861 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4921 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4981 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5041 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5101 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5161 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5221 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5281 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5341 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5401 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5461 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5521 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5581 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5641 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5701 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5761 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5821 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     5881 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     5941 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6001 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6061 tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgtagcggga tca
//
