LOCUS       sequence-361598-        3307 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RMCE vector to shuttle puromycin resistance 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 3307)
  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 3307)
  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-attB53
FEATURES             Location/Qualifiers
     source          1..3307
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      102..188
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      280..307
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     315..338
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    357..456
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    488..521
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             542..1141
                     /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(542..561)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1038..1058
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1222..1239)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1228..1452
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    1478..1511
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     protein_bind    complement(1535..1634)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1746..1765)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1757..2566
                     /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(1824..1843)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2656..3244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3145..3164
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgctagcat ggatctcggg
       61 gacgtctaac tactaagcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc
      121 agtcggaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta
      181 ggacaaatcc gccgggagcg gatttgaacg ttgtgaagca acggcccgga gggtggcggg
      241 caggacgccc gccataaact gccaggcatc aaactaagca gaaggccatc ctgacggatg
      301 gcctttttgc gtttctacaa actcttcctg ttagttagtt acttaagctc gggccccaaa
      361 taatgatttt attttgactg atagtgacct gttcgttgca acaaattgat aagcaatgct
      421 tttttataat gccaactttg tacaaaaaag caggctggcg acggatcggg agatctccgc
      481 ggtgcgggtg ccagggcgtg cccttgggct ccccgggcgc gtactccacc acgccgccac
      541 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
      601 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
      661 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
      721 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
      781 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
      841 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
      901 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
      961 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     1021 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     1081 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     1141 acgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcgat
     1201 gatctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     1261 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     1321 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     1381 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     1441 tgggctctat ggcttctgat atcgtgccgc ggtgcgggtg ccagggcgtg cccttgggct
     1501 ccccgggcgc gtactccact gcgcattaat ctagacccag ctttcttgta caaagttggc
     1561 attataagaa agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat
     1621 aaaatcatta tttgccatcc agctgcagct ctggcccgtg tctcaaaatc tctgatgtta
     1681 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     1741 taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc
     1801 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     1861 tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     1921 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     1981 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     2041 caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     2101 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     2161 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     2221 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     2281 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     2341 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     2401 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     2461 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     2521 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa
     2581 ttggttgtaa cattattcag attgggcccc gttccactga gcgtcagacc ccgtagaaaa
     2641 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2701 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2761 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     2821 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2881 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2941 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3001 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3061 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3121 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3181 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3241 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3301 catgttc
//
