LOCUS       sequence-217854-        6165 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  safe haven knock-in plasmid for the act5 locus, Expression of 
            LifeAct-GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6165)
  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 6165)
  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: pPI227
FEATURES             Location/Qualifiers
     source          1..6165
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             723..773
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     CDS             801..1508
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI
                     CTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNY
                     KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF
                     KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFV
                     TAAGITHGMDELYK"
     primer_bind     complement(825..853)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1448..1468
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    1548..1581
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1855..2892)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
                     MGEAN"
     protein_bind    3328..3361
                     /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(4223..4240)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4394..4982)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4474..4493)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5153..5944,5945..6013))
                     /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     5776..5795
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6014..6118)
                     /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 aaaaaaaaaa tggttttgaa aatttttaaa
      541 aaaatggttg ggtcacccac aatttttata aaaaaaagcc atttggatag tttttttttt
      601 tttttttttt atttttttta tttttttttt tttgagattt ggtataaata caaaaaaaaa
      661 attgttttta aatcattatt aataaaaact taaaataaat tatatataaa aaaagatcca
      721 aaatgggagt agctgattta ataaaaaaat ttgaatcaat atctaaagaa gaaggagatc
      781 cacctgtttc tagtagtggt aaaggagaag aacttttcac tggagttgtc ccaattcttg
      841 ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg
      901 atgcaacata cggaaaactt acccttaaat ttatttgcac tactggaaaa ctacctgttc
      961 catggccaac acttgtcact acttttacgt atggtgttca atgcttttca agatacccag
     1021 atcatatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa
     1081 gaactatatt tttcaaagat gacgggaact acaagacacg tgctgaagtc aagtttgaag
     1141 gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca
     1201 ttcttggaca caaattggaa tacaactata actcacacaa tgtatacatc atggcagaca
     1261 aacaaaagaa tggaatcaaa gttaacttca aaattagaca caacattgaa gatggaagcg
     1321 ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac
     1381 cagacaacca ttacctgtcc acacaatctg ccctttcgaa agatcccaac gaaaagagag
     1441 accacatggt ccttcttgag tttgtaacag ctgctgggat tacacatggc atggatgaac
     1501 tatacaagta aactagtata aaataaataa attgtggatc tgatatcata acttcgtata
     1561 atgtatgcta tacgaagtta tactactctc gagattttat ttaatatact aaataataaa
     1621 aaagttaaaa aatgatcatt ggataaattt tttataatta taaataaaga taataatttt
     1681 ttttttaaca aaactaaaaa taaaaataat aaaataattg ttaaaatagg tttttttttt
     1741 tttttttttt tttaataaat ggtatttatt aatttatttg ttgtgtgtgt ttttttttta
     1801 taatattttt ttttttagca ttgaattaag aagaaatcaa attgatgcgg ccgcttagtt
     1861 agcctccccc atctcccgat ccggacgagt gctggggcgt cggtttccac tatcggcgag
     1921 tacttctaca cagccatcgg tccagacggc cgcgcttctg cgggcgattt gtgtacgccc
     1981 gacagtcccg gctccggatc ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc
     2041 atcgaaattg ccgtcaacca agctctgata gagttggtca agaccaatgc ggagcatata
     2101 cgcccggagc cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg
     2161 ctgctccata caagccaacc acggcctcca gaagaagatg ttggcgacct cgtattggga
     2221 atccccgaac atcgcctcgc tccagtcaat gaccgctgtt atgcggccat tgtccgtcag
     2281 gacattgttg gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc
     2341 ccaaagcatc agctcatcga gagcctgcgc gacggacgca ctgacggtgt cgtccatcac
     2401 agtttgccag tgatacacat ggggatcagc aatcgcgcat atgaaatcac gccatgtagt
     2461 gtattgaccg attccttgcg gtccgaatgg gccgaacccg ctcgtctggc taagatcggc
     2521 cgcagcgatc gcatccatgg cctccgcgac cggctgcaga acagcgggca gttcggtttc
     2581 aggcaggtct tgcaacgtga caccctgtgc acggcgggag atgcaatagg tcaggctctc
     2641 gctgaactcc ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg caaagtgccg
     2701 ataaacataa cgatctttgt agaaaccatc ggcgcagcta tttacccgca ggacatatcc
     2761 acgccctcct acatcgaagc tgaaagcacg agattcttcg ccctccgaga gctgcatcag
     2821 gtcggagacg ctgtcgaact tttcgatcag aaacttctcg acagacgtcg cggtgagttc
     2881 aggctttttc attaggtctt gttgagaaat gttaaattga tccatttttt gctagctgtg
     2941 aaattagttt aaaatacaaa taaagagtta taataatata cagttgaata aaaaaaaaaa
     3001 aaatgaattg gaaaatttat ttttatatga aaaaaaaaaa attttaaaaa aaaaaaaaaa
     3061 aattaaaaaa aaaaaaaaaa aaaaaaaaaa taaaaaaaaa ttaaaatgtg ggtgaaccaa
     3121 attttattta aaaaaaaaaa aaaaatggat cacttttggg tggttggaaa aaaaaaaaaa
     3181 aaaaaattga aaatataatg ttagtcatat gattaatcat taattaattc tttttttttt
     3241 tttttttatt ttttattttt ttttttcaat attccaatta tgacacacac taacatcaca
     3301 aaatacaaaa ttttgggatc cactactata acttcgtata atgtatgcta tacgaagtta
     3361 tttaattaat taagatatca agcttgtcca cagaaaatgt ttctaaacaa aaaaaaaaaa
     3421 ccgagtgatg aaagcgcttc tcacaaaatt atttatgtaa aatatttaat aaaatacatt
     3481 atttaaatct tttttatttt ttttagttgt tgatctttat ccgactttaa aaataaaaaa
     3541 attgtaaaaa aaaaaaaagt ttatttgttt aattttattg ttgtttttaa ttttttacaa
     3601 ccattaattt ataaaaccaa cacctaacta taatgaaata caatttgtat cttggaaaaa
     3661 aaaaaattaa taatataaat aaaatgtaaa caagtatccg gtggaaggaa ctaacagttg
     3721 ccttatccag ttcaaaaaaa aaaaaaaaaa aagaaattaa attaccattt tatatttaac
     3781 taaaataaat taaaaggttt attttaataa tagatttatt attttttaaa aatcaataat
     3841 taatgttgga taagatgaac gaataaaaga tagagtttca ggtcttttaa atgatgagga
     3901 tttatgaatt cttaaaagtt ttgggcaaga gaaaatgatt ttaacgattg aaccatcact
     3961 aattgcaaaa tctcttgaac taagagtttt taagtttgaa gcaagtggag aattacaaag
     4021 agcaatcata gagttatcag ttaaatcata acaacaagat aaagataaag aactaatatg
     4081 atgtaaattt tcaatcatac ctctaaaaga aacatcattc atactttttg gataaattaa
     4141 atctaatgaa tctaaatttg gaaatagtaa accaatattt ggccggcaga ggcggtttgc
     4201 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4261 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4321 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4381 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4441 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4501 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4561 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4621 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4681 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4741 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4801 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4861 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4921 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4981 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5041 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5101 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5161 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5221 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5281 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5341 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5401 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5461 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5521 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5581 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5641 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5701 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5761 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5821 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5881 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5941 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6001 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6061 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6121 catttccccg aaaagtgcca cctgacgcgc cctgtagcgg gatca
//
