LOCUS       sequence-414403-        6107 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Safe haven knock-in plasmid for the act5 locus for C-terminal 
            mNeonGreen fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6107)
  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 6107)
  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: pPI230
FEATURES             Location/Qualifiers
     source          1..6107
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1490..1523
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1797..2834)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
                     MGEAN"
     protein_bind    3270..3303
                     /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(4165..4182)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4336..4924)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4416..4435)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5095..5886,5887..5955))
                     /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     5718..5737
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5956..6060)
                     /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 gkttttkaaa atttttaaaa
      541 aaatgkttgg ktyacccama attyttataa aaaaaakcya tttggatagt tttttttttt
      601 ttttttttta ttttttttat tttttttttt ttgagatttg gtataaatac aaaaaaaaaa
      661 ttgtttttaa atcattatta ataaaaactt aaaataaatt atatataaaa aaagatcttc
      721 aactagtggt ggttcaggag gtagtgtgag taaaggtgaa gaagataata tggcatcgtt
      781 accagctaca catgagttac atatattcgg tagcattaat ggtgttgatt ttgatatggt
      841 gggacaaggt accggtaatc ctaatgatgg ttacgaagaa ctaaatttaa aatcgactaa
      901 aggtgactta caattttctc catggatttt agtgccacat atagggtatg gttttcatca
      961 atacttacca tatccagatg gtatgtcacc atttcaagct gcaatggttg atggatcagg
     1021 ttatcaagtt catagaacaa tgcaatttga agatggtgct tcattaactg ttaattatag
     1081 atacacatat gaaggctcac atattaaagg tgaagctcaa gttaaaggta ctggtttccc
     1141 agccgatggc ccagttatga caaatagttt aacagcagca gattggtgta gatccaaaaa
     1201 aacttatcca aatgataaaa caattatttc aacttttaaa tggtcatata caaccggtaa
     1261 tggtaaacgt tatcgttcaa cagcccgtac aacatatact tttgctaaac caatggcagc
     1321 taattattta aaaaatcaac caatgtatgt ttttcgtaaa acagagttaa aacattcaaa
     1381 aacagaactt aattttaaag aatggcaaaa agcatttaca gacgttatgg gtatggatga
     1441 actttataag taagctagta taaaataaat aaattgtgga tctgatatca taacttcgta
     1501 taatgtatgc tatacgaagt tatactactc tcgagatttt atttaatata ctaaataata
     1561 aaaaagttaa aaaatgatca ttggataaat tttttataat tataaataaa gataataatt
     1621 ttttttttaa caaaactaaa aataaaaata ataaaataat tgttaaaata ggtttttttt
     1681 tttttttttt tttttaataa atggtattta ttaatttatt tgttgtgtgt gttttttttt
     1741 tataatattt ttttttttag cattgaatta agaagaaatc aaattgatgc ggccgcttag
     1801 ttagcctccc ccatctcccg atccggacga gtgctggggc gtcggtttcc actatcggcg
     1861 agtacttcta cacagccatc ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc
     1921 ccgacagtcc cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca
     1981 tcatcgaaat tgccgtcaac caagctctga tagagttggt caagaccaat gcggagcata
     2041 tacgcccgga gccgcggcga tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc
     2101 tgctgctcca tacaagccaa ccacggcctc cagaagaaga tgttggcgac ctcgtattgg
     2161 gaatccccga acatcgcctc gctccagtca atgaccgctg ttatgcggcc attgtccgtc
     2221 aggacattgt tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg
     2281 gcccaaagca tcagctcatc gagagcctgc gcgacggacg cactgacggt gtcgtccatc
     2341 acagtttgcc agtgatacac atggggatca gcaatcgcgc atatgaaatc acgccatgta
     2401 gtgtattgac cgattccttg cggtccgaat gggccgaacc cgctcgtctg gctaagatcg
     2461 gccgcagcga tcgcatccat ggcctccgcg accggctgca gaacagcggg cagttcggtt
     2521 tcaggcaggt cttgcaacgt gacaccctgt gcacggcggg agatgcaata ggtcaggctc
     2581 tcgctgaact ccccaatgtc aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc
     2641 cgataaacat aacgatcttt gtagaaacca tcggcgcagc tatttacccg caggacatat
     2701 ccacgccctc ctacatcgaa gctgaaagca cgagattctt cgccctccga gagctgcatc
     2761 aggtcggaga cgctgtcgaa cttttcgatc agaaacttct cgacagacgt cgcggtgagt
     2821 tcaggctttt tcattaggtc ttgttgagaa atgttaaatt gatccatttt ttgctagctg
     2881 tgaaattagt ttaaaataca aataaagagt tataataata tacagttgaa waaaaaaaaa
     2941 aaaaawraat trraaaattt atttttawaw aaaaaaaaaa aaawtttaaa aaaaaaaaaa
     3001 aaaawwaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aattmaactg tgggtgaccc
     3061 aaattttatt taaaaaaaaa aaaaaaatgg atcacttttg ggtggttgga aaaaaaaaaa
     3121 aaaaaaaatt gaaaatataa tgttagtcat atgattaatc attaattaat tytttttttt
     3181 ttttttttta ttttttattt ttttttttca atattccaat tatgacacac actaacatca
     3241 caaaatacaa aattttggga tccactacta taacttcgta taatgtatgc tatacgaagt
     3301 tatttaatta attaagatat caagcttgtc cacagaaaat gtttctaaac aaaaaaaaaa
     3361 aaccgagtga tgaaagcgct tctcacaaaa ttatttatgt aaaatattta ataaaataca
     3421 ttatttaaat cttttttatt ttttttagtt gttgatcttt atccgacttt aaaaataaaa
     3481 aaattgtaaa aaaaaaaaaa gtttatttgt ttaattttat tgttgttttt aattttttac
     3541 aaccattaat ttataaaacc aacacctaac tataatgaaa tacaatttgt atcttggaaa
     3601 aaaaaaaatt aataatataa ataaaatgta aacaagtatc cggtggaagg aactaacagt
     3661 tgccttatcc agttcaaaaa aaaaaaaaaa aaaagaaatt aaattaccat tttatattta
     3721 actaaaataa attaaaaggt ttattttaat aatagattta ttatttttta aaaatcaata
     3781 attaatgttg gataagatga acgaataaaa gatagagttt caggtctttt aaatgatgag
     3841 gatttatgaa ttcttaaaag ttttgggcaa gagaaaatga ttttaacgat tgaaccatca
     3901 ctaattgcaa aatctcttga actaagagtt tttaagtttg aagcaagtgg agaattacaa
     3961 agagcaatca tagagttatc agttaaatca taacaacaag ataaagataa agaactaata
     4021 tgatgtaaat tttcaatcat acctctaaaa gaaacatcat tcatactttt tggataaatt
     4081 aaatctaatg aatctaaatt tggaaatagt aaaccaatat ttggccggca gaggcggttt
     4141 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4201 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4261 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4321 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4381 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4441 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4501 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4561 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4621 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4681 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4741 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4801 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4861 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4921 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4981 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5041 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5101 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5161 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5221 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5281 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5341 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5401 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5461 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5521 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5581 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5641 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5701 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5761 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5821 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5881 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5941 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6001 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6061 cacatttccc cgaaaagtgc cacctgacgc gccctgtagc gggatca
//
