LOCUS       sequence-217736-        6120 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  safe haven knock-in plasmid for the act5 locus, N-terminal 
            mTurquoise2 tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6120)
  TITLE     Kay lab plasmids
REFERENCE   2  (bases 1 to 6120)
  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"
COMMENT     Sequence Label: pPI228
FEATURES             Location/Qualifiers
     source          1..6120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1504..1537
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1810..2847)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
                     MGEAN"
     protein_bind    3283..3316
                     /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(4178..4195)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4349..4937)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4429..4448)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5108..5899,5900..5968))
                     /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     5731..5750
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5969..6073)
                     /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 gtcacccaca atttttataa aaaaaagcca tttggatagt tttttttttt
      601 ttttttttta ttttttttat tttttttttt ttgagatttg gtataaatac aaaaaaaaaa
      661 ttgtttttaa atcattatta ataaaaactt aaaataaatt atatataaaa aaagatccaa
      721 aaaatggtta gtaaaggtga ggaattattt acaggtgttg ttccaatatt agtagaactt
      781 gatggtgatg taaacggtca taaattttca gtctcaggtg aaggtgaagg tgatgcaaca
      841 tacggaaaat taaccctcaa atttatttgt actactggta aattacctgt tccatggcca
      901 actttggtaa ctacactaag ttggggcgtt caatgttttg cgagatatcc agatcatatg
      961 aaacaacatg atttctttaa gtcagctatg ccagaaggat atgtacaaga aagaacaata
     1021 ttttttaagg atgatgggaa ttataagaca agagccgaag ttaaatttga aggtgatacc
     1081 ttagtaaaca gaattgaatt aaaaggtatt gatttcaaag aggatggaaa tatattaggt
     1141 cacaaattag agtataatta tttctcagat aatgtatata taacagccga caaacaaaaa
     1201 aatggaataa aagctaactt taaaatccgt cataatatag aggatggagg agttcaatta
     1261 gccgatcatt atcaacaaaa tactccaatt ggagatggtc cagttttgtt accagataat
     1321 cactatttat caactcaatc taaattgagt aaagatccta atgagaaaag agatcatatg
     1381 gttttgttgg agttcgttac tgcagcaggt attactttag gtatggatga attatataaa
     1441 ggtggttcag gaggtagtag atcttcaact agtataaaat aaataaattg tggatctgat
     1501 atcataactt cgtataatgt atgctatacg aagttatact actctcgaga ttttatttaa
     1561 tatactaaat aataaaaaag ttaaaaaatg atcattggat aaatttttta taattataaa
     1621 taaagataat aatttttttt ttaacaaaac taaaaataaa aataataaaa taattgttaa
     1681 aataggtttt tttttttttt ttttttttaa taaatggtat ttattaattt atttgttgtg
     1741 tgtgtttttt ttttataata tttttttttt tagcattgaa ttaagaagaa atcaaattga
     1801 tgcggccgct tagttagcct cccccatctc ccgatccgga cgagtgctgg ggcgtcggtt
     1861 tccactatcg gcgagtactt ctacacagcc atcggtccag acggccgcgc ttctgcgggc
     1921 gatttgtgta cgcccgacag tcccggctcc ggatcggacg attgcgtcgc atcgaccctg
     1981 cgcccaagct gcatcatcga aattgccgtc aaccaagctc tgatagagtt ggtcaagacc
     2041 aatgcggagc atatacgccc ggagccgcgg cgatcctgca agctccggat gcctccgctc
     2101 gaagtagcgc gtctgctgct ccatacaagc caaccacggc ctccagaaga agatgttggc
     2161 gacctcgtat tgggaatccc cgaacatcgc ctcgctccag tcaatgaccg ctgttatgcg
     2221 gccattgtcc gtcaggacat tgttggagcc gaaatccgcg tgcacgaggt gccggacttc
     2281 ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc tgcgcgacgg acgcactgac
     2341 ggtgtcgtcc atcacagttt gccagtgata cacatgggga tcagcaatcg cgcatatgaa
     2401 atcacgccat gtagtgtatt gaccgattcc ttgcggtccg aatgggccga acccgctcgt
     2461 ctggctaaga tcggccgcag cgatcgcatc catggcctcc gcgaccggct gcagaacagc
     2521 gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc tgtgcacggc gggagatgca
     2581 ataggtcagg ctctcgctga actccccaat gtcaagcact tccggaatcg ggagcgcggc
     2641 cgatgcaaag tgccgataaa cataacgatc tttgtagaaa ccatcggcgc agctatttac
     2701 ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa gcacgagatt cttcgccctc
     2761 cgagagctgc atcaggtcgg agacgctgtc gaacttttcg atcagaaact tctcgacaga
     2821 cgtcgcggtg agttcaggct ttttcattag gtcttgttga gaaatgttaa attgatccat
     2881 tttttgctag ctgtgaaatt agtttaaaat acaaataaag agttataata atatacagtt
     2941 gaataaaaaa aaaaaaaatg aattggaaaa tttattttta tataaaaaaa aaaaaatttt
     3001 aaaaaaaaaa aaaaaaatta aaaaaaaaaa aaaaaaaaaa aaaaaataaa aaaaattaaa
     3061 atgtgggtga accaaatttt atttaaaaaa aaaaaaaaaa tggatcactt ttgggtggtt
     3121 ggaaaaaaaa aaaaaaaaaa attgaaaata taatgttagt catatgatta atcattaatt
     3181 aattcttttt tttttttttt ttatttttta tttttttttt tcaatattcc aattatgaca
     3241 cacactaaca tcacaaaata caaaattttg ggatccacta ctataacttc gtataatgta
     3301 tgctatacga agttatttaa ttaattaaga tatcaagctt gtccacagaa aatgtttcta
     3361 aacaaaaaaa aaaaaccgag tgatgaaagc gcttctcaca aaattattta tgtaaaatat
     3421 ttaataaaat acattattta aatctttttt atttttttta gttgttgatc tttatccgac
     3481 tttaaaaata aaaaaattgt aaaaaaaaaa aaagtttatt tgtttaattt tattgttgtt
     3541 tttaattttt tacaaccatt aatttataaa accaacacct aactataatg aaatacaatt
     3601 tgtatcttgg aaaaaaaaaa attaataata taaataaaat gtaaacaagt atccggtgga
     3661 aggaactaac agttgcctta tccagttcaa aaaaaaaaaa aaaaaaagaa attaaattac
     3721 cattttatat ttaactaaaa taaattaaaa ggtttatttt aataatagat ttattatttt
     3781 ttaaaaatca ataattaatg ttggataaga tgaacgaata aaagatagag tttcaggtct
     3841 tttaaatgat gaggatttat gaattcttaa aagttttggg caagagaaaa tgattttaac
     3901 gattgaacca tcactaattg caaaatctct tgaactaaga gtttttaagt ttgaagcaag
     3961 tggagaatta caaagagcaa tcatagagtt atcagttaaa tcataacaac aagataaaga
     4021 taaagaacta atatgatgta aattttcaat catacctcta aaagaaacat cattcatact
     4081 ttttggataa attaaatcta atgaatctaa atttggaaat agtaaaccaa tatttggccg
     4141 gcagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4201 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4261 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4321 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4381 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4441 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4501 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4561 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4621 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4681 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4741 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4801 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4861 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4921 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4981 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5041 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5101 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5161 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5221 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5281 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5341 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5401 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5461 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5521 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5581 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5641 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5701 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5761 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5821 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5881 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5941 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6001 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6061 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt agcgggatca
//
