LOCUS       sequence-361601-        5107 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RMCE vector to shuttle puromycin resistance and tetO-mCherry 
            cassettes into attP50-flanked landing pad. Designed for use with 
            pmROSA26-attP50-Neo-mKate2-3xNLS-attP50 vector (Addgene 183609)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5107)
  AUTHORS   Malaguti M, Migueles RP, Annoh J, Sadurska D, Blin G, Lowell S
  TITLE     SyNPL: Synthetic notch pluripotent cell lines to monitor and 
            manipulate cell interactions in vitro and in vivo.
  JOURNAL   Development. 2022 May 26. pii: 275525. doi: 10.1242/dev.200226.
  PUBMED    35616331
REFERENCE   2  (bases 1 to 5107)
  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: 
            "Development. 2022 May 26. pii: 275525. doi: 10.1242/dev.200226."
COMMENT     Sequence Label: attB53-Pac-tetO-mCherry-attB53
FEATURES             Location/Qualifiers
     source          1..5107
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      100..186
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      278..305
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     313..336
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    355..454
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    486..519
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             540..1139
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(540..559)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1036..1056
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1220..1237)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1226..1450
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1468..1484
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1468..1484
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1827..1846
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    1845..1900
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     1946..1965
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(2073..2783)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2351..2370)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2620..2638
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2780..2789
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2881..2905)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    2927..2945
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2964..2982
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3001..3019
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3038..3056
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3075..3093
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3112..3130
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3149..3167
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3186..3204
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3276..3309
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     protein_bind    complement(3333..3432)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3544..3563)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3555..4364
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3622..3641)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4454..5042
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4943..4962
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gctagcatgg atctcgggga
       61 cgtctaacta ctaagcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
      121 tcggaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
      181 acaaatccgc cgggagcgga tttgaacgtt gtgaagcaac ggcccggagg gtggcgggca
      241 ggacgcccgc cataaactgc caggcatcaa actaagcaga aggccatcct gacggatggc
      301 ctttttgcgt ttctacaaac tcttcctgtt agttagttac ttaagctcgg gccccaaata
      361 atgattttat tttgactgat agtgacctgt tcgttgcaac aaattgataa gcaatgcttt
      421 tttataatgc caactttgta caaaaaagca ggctggcgac ggatcgggag atctccgcgg
      481 tgcgggtgcc agggcgtgcc cttgggctcc ccgggcgcgt actccaccac gccgccacca
      541 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
      601 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
      661 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
      721 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
      781 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
      841 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
      901 ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca
      961 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     1021 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     1081 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac
     1141 gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc acgaccccat gcatcgatga
     1201 tctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     1261 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     1321 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     1381 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     1441 ggctctatgg cttctgatat cggatcccag gaaacagcta tgaccatgat tacgccaagc
     1501 ttgggctgca ggtcgaggga tctccataag agaagaggga cagctatgac tgggagtagt
     1561 caggagagga ggaaaaatct ggctagtaaa acatgtaagg aaaattttag ggatgttaaa
     1621 gaaaaaaata acacaaaaca aaatataaaa aaaatctaac ctcaagtcaa ggcttttcta
     1681 tggaataagg aatggacagc agggggctgt ttcatatact gatgacctct ttatagccaa
     1741 cctttgttca tggcagccag catatgggca tatgttgcca aactctaaac caaatactca
     1801 ttctgatgtt ttaaatgatt tgccctccca tatgtccttc cgagtgagag acacaaaaaa
     1861 ttccaacaca ctattgcaat gaaaataaat ttcctttatt agccagaagt cagatgctca
     1921 aggggcttca tgatgtcccc ataatttttg gcagagggaa aaagatctca gtggtatttg
     1981 tgagccagtt ggccacacca gccaccacct tctgataggc agcctgcacc tgaggagtga
     2041 attcgatatc aagcttatcg agcggccgct catcacttgt acagctcgtc catgccgccg
     2101 gtggagtggc ggccctcggc gcgttcgtac tgttccacga tggtgtagtc ctcgttgtgg
     2161 gaggtgatgt ccaacttgat gttgacgttg taggcgccgg gcagctgcac gggcttcttg
     2221 gccttgtagg tggtcttgac ctcagcgtcg tagtggccgc cgtccttcag cttcagcctc
     2281 tgcttgatct cgcccttcag ggcgccgtcc tcggggtaca tccgctcgga ggaggcctcc
     2341 cagcccatgg tcttcttctg cattacgggg ccgtcggagg ggaagttggt gccgcgcagc
     2401 ttcaccttgt agatgaactc gccgtcctgc agggaggagt cctgggtcac ggtcaccacg
     2461 ccgccgtcct cgaagttcat cacgcgctcc cacttgaagc cctcggggaa ggacagcttc
     2521 aagtagtcgg ggatgtcggc ggggtgcttc acgtaggcct tggagccgta catgaactga
     2581 ggggacagga tgtcccaggc gaagggcagg gggccaccct tggtcacctt cagcttggcg
     2641 gtctgggtgc cctcgtaggg gcggccctcg ccctcgccct cgatctcgaa ctcgtggccg
     2701 ttcacggagc cctccatgtg caccttgaag cgcatgaact ccttgatgat ggccatgtta
     2761 tcctcctcgc ccttgctcac catggtggcg gcctcgagtg gcgcgccggc cggcccggag
     2821 gctggatcgg tcccggtgtc ttctatggag gtcaaaacag cgtggatggc gtctccaggc
     2881 gatctgacgg ttcactaaac gagctctgct tatatagggg gtaccttctc tatcactgat
     2941 agggagctcg agacgtgtcg acttctctat cactgatagg gagacgttct agacgtacct
     3001 tctctatcac tgatagggag ctcgagacgt gtcgacttct ctatcactga tagggagacg
     3061 ttctagacgt accttctcta tcactgatag ggagctcgag acgtgtcgac ttctctatca
     3121 ctgataggga gacgttctag acgtaccttc tctatcactg atagggagct cgagacgtgt
     3181 cgacttctct atcactgata gggagacgtg cggccagctt ggatcactac gcgccagtgc
     3241 tccgagacgg aattcgatat cgtgccgcgg tgcgggtgcc agggcgtgcc cttgggctcc
     3301 ccgggcgcgt actccactgc gcattaatct agacccagct ttcttgtaca aagttggcat
     3361 tataagaaag cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa
     3421 aatcattatt tgccatccag ctgcagctct ggcccgtgtc tcaaaatctc tgatgttaca
     3481 ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta
     3541 atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca
     3601 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     3661 cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     3721 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     3781 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     3841 ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     3901 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     3961 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     4021 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     4081 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     4141 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     4201 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     4261 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     4321 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt
     4381 ggttgtaaca ttattcagat tgggccccgt tccactgagc gtcagacccc gtagaaaaga
     4441 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4501 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4561 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4621 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4681 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4741 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4801 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4861 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4921 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4981 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5041 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5101 tgttctt
//
