LOCUS       sequence-414402-        6111 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Safe haven knock-in plasmid for the act5 locus for N-terminal 
            mNeonGreen fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6111)
  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 6111)
  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: pPI231
FEATURES             Location/Qualifiers
     source          1..6111
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1494..1527
                     /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    3274..3307
                     /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(4169..4186)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4340..4928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4420..4439)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5099..5890,5891..5959))
                     /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     5722..5741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5960..6064)
                     /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 aaaaaaaatg kttttkaaaa tttttaaaaa
      541 aatgkttggk tmaccmamaa ttyttataaa aaaaakyyat ttggatagtt tttttttttt
      601 ttttttttat tttttttatt tttttttttt tgagatttgg tataaataca aaaaaaaaat
      661 tgtttttaaa tcattattaa taaaaactta aaataaatta tatataaaaa aagatccaaa
      721 aaatggtgag taaaggtgaa gaagataata tggcatcgtt accagctaca catgagttac
      781 atatattcgg tagcattaat ggtgttgatt ttgatatggt gggacaaggt accggtaatc
      841 ctaatgatgg ttacgaagaa ctaaatttaa aatcgactaa aggtgactta caattttctc
      901 catggatttt agtgccacat atagggtatg gttttcatca atacttacca tatccagatg
      961 gtatgtcacc atttcaagct gcaatggttg atggatcagg ttatcaagtt catagaacaa
     1021 tgcaatttga agatggtgct tcattaactg ttaattatag atacacatat gaaggctcac
     1081 atattaaagg tgaagctcaa gttaaaggta ctggtttccc agccgatggc ccagttatga
     1141 caaatagttt aacagcagca gattggtgta gatccaaaaa aacttatcca aatgataaaa
     1201 caattatttc aacttttaaa tggtcatata caaccggtaa tggtaaacgt tatcgttcaa
     1261 cagcccgtac aacatatact tttgctaaac caatggcagc taattattta aaaaatcaac
     1321 caatgtatgt ttttcgtaaa acagagttaa aacattcaaa aacagaactt aattttaaag
     1381 aatggcaaaa agcatttaca gacgttatgg gtatggatga actttataag ggtggttcag
     1441 gaggtagtag atcttcaact agtataaaat aaataaattg tggatctgat atcataactt
     1501 cgtataatgt atgctatacg aagttatact actctcgaga ttttatttaa tatactaaat
     1561 aataaaaaag ttaaaaaatg atcattggat aaatttttta taattataaa taaagataat
     1621 aatttttttt ttaacaaaac taaaaataaa aataataaaa taattgttaa aataggtttt
     1681 tttttttttt ttttttttta ataaatggta tttattaatt tatttkttgt gtgtkttttt
     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 aaaaaaaaaw raattrraaa atttattttt awawaaaaaa aaaaaaawtt traaaaaaaa
     3001 aaaaaaaaww aaaaaaaaaa aaaaaaaaaa aaaaaawaaa aaaaaattma actgtgggtr
     3061 acccaaattt tatttaaaaa aaaaaaaaaa atggatcact tttgggtggt tkgaaaaaaa
     3121 aaaaaaaaaa aattgaaaat ataatgttag tcatatgatt aatcattaat taattytttt
     3181 tttttttttt tttatttttt attttttttt ttcaatattc caattatgac acacactaac
     3241 atcacaaaat acaaaatttt gggatccact actataactt cgtataatgt atgctatacg
     3301 aagttattta attaattaag atatcaagct tgtccacaga aaatgtttct aaacaaaaaa
     3361 aaaaaaccga gtgatgaaag cgcttctcac aaaattattt atgtaaaata tttaataaaa
     3421 tacattattt aaatcttttt tatttttttt agttgttgat ctttatccga ctttaaaaat
     3481 aaaaaaattg taaaaaaaaa aaaagtttat ttgtttaatt ttattgttgt ttttaatttt
     3541 ttacaaccat taatttataa aaccaacacc taactataat gaaatacaat ttgtatcttg
     3601 gaaaaaaaaa aattaataat ataaataaaa tgtaaacaag tatccggtgg aaggaactaa
     3661 cagttgcctt atccagttca aaaaaaaaaa aaaaaaaaga aattaaatta ccattttata
     3721 tttaactaaa ataaattaaa aggtttattt taataataga tttattattt tttaaaaatc
     3781 aataattaat gttggataag atgaacgaat aaaagataga gtttcaggtc ttttaaatga
     3841 tgaggattta tgaattctta aaagttttgg gcaagagaaa atgattttaa cgattgaacc
     3901 atcactaatt gcaaaatctc ttgaactaag agtttttaag tttgaagcaa gtggagaatt
     3961 acaaagagca atcatagagt tatcagttaa atcataacaa caagataaag ataaagaact
     4021 aatatgatgt aaattttcaa tcatacctct aaaagaaaca tcattcatac tttttggata
     4081 aattaaatct aatgaatcta aatttggaaa tagtaaacca atatttggcc ggcagaggcg
     4141 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4201 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4261 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4321 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4381 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4441 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4501 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4561 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4621 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4681 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4741 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4801 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4861 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4921 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4981 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5041 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5101 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5161 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5221 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5281 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5341 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5401 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5461 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5521 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5581 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5641 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5701 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5761 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5821 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5881 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5941 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6001 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6061 cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg tagcgggatc a
//
