LOCUS       sequence-205381-        6112 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  safe haven knock-in plasmid for the act5 locus, C-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: pDM1516
FEATURES             Location/Qualifiers
     source          1..6112
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1492..1525
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1801..2838)
                     /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 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 aggtagtgtt tcaaaaggtg aagaagataa tatggcaatt
      781 attaaagaat ttatgagatt taaagttcat atggaaggtt cagttaatgg tcatgaattt
      841 gaaattgaag gtgaaggtga aggtagacca tatgaaggta cacaaacagc aaaattaaaa
      901 gttacaaaag gtggtccatt accatttgca tgggatattt tatcaccaca atttatgtat
      961 ggttcaaaag catatgttaa acatccagca gatattccag attatttaaa attatcattt
     1021 ccagaaggtt ttaaatggga aagagttatg aattttgaag atggtggtgt tgttacagtt
     1081 acacaagatt catcattaca agatggtgaa tttatttata aagttaaatt aagaggtaca
     1141 aattttccat cagatggtcc agttatgcaa aagaaaacaa tgggttggga agcatcatca
     1201 gaaagaatgt atccagaaga tggtgcatta aaaggtgaaa ttaaacaaag attaaaatta
     1261 aaagatggtg gtcattatga tgcagaagtt aaaacaacat ataaagcaaa aaaaccagtt
     1321 caattaccag gtgcatataa tgttaatatt aaattagata ttacatcaca taatgaagat
     1381 tatacaattg ttgaacaata tgaaagagca gaaggtagac attcaacagg tggtatggat
     1441 gaattatata aataagctag tataaaataa ataaattgtg gatctgatat cataacttcg
     1501 tataatgtat gctatacgaa gttatactac tctcgagatt ttatttaata tactaaataa
     1561 taaaaaagtt aaaaaatgat cattggataa attttttata attataaata aagataataa
     1621 tttttttttt aacaaaacta aaaataaaaa taataaaata attgttaaaa taggtttttt
     1681 tttttttttt ttttttttaa taaatggtat ttattaattt atttgttgtg tgtgtttttt
     1741 tttttataat attttttttt ttagcattga attaagaaga aatcaaattg atgcggccgc
     1801 ttagttagcc tcccccatct cccgatccgg acgagtgctg gggcgtcggt ttccactatc
     1861 ggcgagtact tctacacagc catcggtcca gacggccgcg cttctgcggg cgatttgtgt
     1921 acgcccgaca gtcccggctc cggatcggac gattgcgtcg catcgaccct gcgcccaagc
     1981 tgcatcatcg aaattgccgt caaccaagct ctgatagagt tggtcaagac caatgcggag
     2041 catatacgcc cggagccgcg gcgatcctgc aagctccgga tgcctccgct cgaagtagcg
     2101 cgtctgctgc tccatacaag ccaaccacgg cctccagaag aagatgttgg cgacctcgta
     2161 ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc
     2221 cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc
     2281 ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc
     2341 catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca
     2401 tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag
     2461 atcggccgca gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag cgggcagttc
     2521 ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag
     2581 gctctcgctg aactccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa
     2641 gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac
     2701 atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg
     2761 catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt
     2821 gagttcaggc tttttcatta ggtcttgttg agaaatgtta aattgatcca ttttttgcta
     2881 gctgtgaaat tagtttaaaa tacaaataaa gagttataat aatatacagt tgaataaaaa
     2941 aaaaaaaaat gaattggaaa atttattttt atatgaagaa aaaaaaattt tgaaaaaaaa
     3001 aaaaaaaatt aaaaaaaaaa aaaaaaaaaa aaaaaattaa aaataattca actgtgggtg
     3061 acccaaattt tatttaaaaa aaaaaaaaaa atggatcact tttgggtggt tggaaaaaaa
     3121 aaaaaaaaaa aattgaaaat ataatgttag tcatatgatt aatcattaat taattctttt
     3181 tttttttttt tttatttttt attttttttt 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 ggacagtatt 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
//
