LOCUS       sequence-361600-        4553 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RMCE vector to shuttle puromycin resistance and TRE-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 4553)
  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 4553)
  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-TRE-mCherry-attB53
FEATURES             Location/Qualifiers
     source          1..4553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      96..182
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      274..301
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     309..332
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    351..450
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    482..515
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             536..1135
                     /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(536..555)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1032..1052
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1216..1233)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1222..1446
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    1474..1595
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1485..1504)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1539..1558
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(2339..2653)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     primer_bind     complement(2342..2366)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    2413..2431
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2449..2467
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2484..2502
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2520..2538
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2556..2574
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2591..2609
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2627..2645
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2718..2751
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     protein_bind    complement(2775..2874)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2986..3005)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2997..3806
                     /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(3064..3083)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3896..4484
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4385..4404
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcgttatccc ctgattctgt ggataaccgt attaccgcta gcatggatct cggggacgtc
       61 taactactaa gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcgg
      121 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa
      181 atccgccggg agcggatttg aacgttgtga agcaacggcc cggagggtgg cgggcaggac
      241 gcccgccata aactgccagg catcaaacta agcagaaggc catcctgacg gatggccttt
      301 ttgcgtttct acaaactctt cctgttagtt agttacttaa gctcgggccc caaataatga
      361 ttttattttg actgatagtg acctgttcgt tgcaacaaat tgataagcaa tgctttttta
      421 taatgccaac tttgtacaaa aaagcaggct ggcgacggat cgggagatct ccgcggtgcg
      481 ggtgccaggg cgtgcccttg ggctccccgg gcgcgtactc caccacgccg ccaccatgac
      541 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
      601 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
      661 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
      721 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
      781 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
      841 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
      901 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
      961 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     1021 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     1081 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacgccc
     1141 gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga ccccatgcat cgatgatcta
     1201 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     1261 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     1321 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     1381 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     1441 ctatggcttc tgatatcgga tccgcagaca tgataagata cattgatgag tttggacaaa
     1501 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     1561 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc tatttgtaca
     1621 gctcgtccat gccgccggtg ctgtgtctgc cctcggctct ctcgtactgc tccacgatgg
     1681 tgtagtcctc gttgtggctg gtgatgtcca gcttgatgtt cacgttgtag gcgccaggca
     1741 gctgcacggg tttcttggcc ttgtaggtgg ttttcacttc ggcgtcgtag tggccgccat
     1801 ccttcagctt cagccgctgc ttgatctcgc ccttcagggc gccatcctcg gggtacattc
     1861 tctcgctgct ggcctcccag cccatggtct ttttctgcat cacggggccg tcgctaggga
     1921 agttggtgcc ccgcagcttc actttgtaga tgaactcgcc gtcctgcagg ctgctgtcct
     1981 gggtcacggt gaccacgccg ccgtcctcga agttcatcac tctctcccac ttgaagccct
     2041 cggggaagct cagcttcagg tagtcgggga tgtcggcggg gtgcttcacg taggccttgc
     2101 tgccgtacat gaactggggg ctcaggatgt cccaggcaaa gggcaggggt ccgcccttgg
     2161 tcactttcag cttggcggtc tgggtgccct cgtaaggtct gccttcgccc tcgccctcga
     2221 tctcgaactc gtggccgttc acgctgcctt ccatgtgcac cttgaaccgc atgaattctt
     2281 tgatgatggc catgttgtcc tcttcgccct tggacaccat ggtgaacata tgtctccagg
     2341 cgatctgacg gttcactaaa cgagctctgc ttatataggc ctcccaccgt acacgcctac
     2401 ctcgacatac gttctctatc actgataggg agtaaactcg acatacgttc tctatcactg
     2461 atagggataa actcgacata cgttctctat cactgatagg gagtaaactc gacatacgtt
     2521 ctctatcact gatagggagt aaactcgaca tacgttctct atcactgata gggagtaaac
     2581 tcgacatcgt tctctatcac tgatagggag taaactcgac atacgttctc tatcactgat
     2641 agggagtaaa ctcgacatat cgattcgaag gcgcgccagt gctccgagac ggaattcgat
     2701 atcgtgccgc ggtgcgggtg ccagggcgtg cccttgggct ccccgggcgc gtactccact
     2761 gcgcattaat ctagacccag ctttcttgta caaagttggc attataagaa agcattgctt
     2821 atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc
     2881 agctgcagct ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata
     2941 tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     3001 gccatattca acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat
     3061 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt
     3121 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     3181 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     3241 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggaa
     3301 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     3361 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     3421 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     3481 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     3541 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     3601 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     3661 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     3721 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     3781 atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa cattattcag
     3841 attgggcccc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3901 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3961 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4021 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4081 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4141 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4201 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4261 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4321 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4381 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4441 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4501 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cct
//
