LOCUS       sequence-361602-        4667 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RMCE vector to shuttle puromycin resistance and TRE-3xFlag-Neurog1 
            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 4667)
  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 4667)
  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-3xFlag-Neurog1-attB53
FEATURES             Location/Qualifiers
     source          1..4667
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(55..643)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(135..154)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(733..1542)
                     /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     1456..1475
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1534..1553
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    1665..1764
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    1788..1821
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        1886..2200
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1894..1912
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1930..1948
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1965..1983
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2001..2019
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2037..2055
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2072..2090
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2108..2126
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2173..2197
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2216..2225
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2225..2290
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    3058..3179
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3095..3114)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3149..3168
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3207..3431
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3420..3437
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3518..4117)
                     /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(3601..3621)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4098..4117
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     protein_bind    4138..4171
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     protein_bind    complement(4203..4302)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4321..4344)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(4352..4379)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4471..4557)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
       61 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
      121 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
      181 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
      241 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
      301 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
      361 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
      421 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
      481 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
      541 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
      601 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
      661 ttctacgggg tctgacgctc agtggaacgg ggcccaatct gaataatgtt acaaccaatt
      721 aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
      781 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
      841 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
      901 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
      961 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     1021 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     1081 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     1141 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     1201 tgaatcagga tattcttcta atacctggaa tgctgttttt ccggggatcg cagtggtgag
     1261 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     1321 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     1381 atgtttcaga aacaactctg gcgcatcggg cttcccatac aagcgataga ttgtcgcacc
     1441 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     1501 atttaatcgc ggcctcgacg tttcccgttg aatatggctc ataacacccc ttgtattact
     1561 gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta
     1621 acatcagaga ttttgagaca cgggccagag ctgcagctgg atggcaaata atgattttat
     1681 tttgactgat agtgacctgt tcgttgcaac aaattgataa gcaatgcttt cttataatgc
     1741 caactttgta caagaaagct gggtctagat taatgcgcag tggagtacgc gcccggggag
     1801 cccaagggca cgccctggca cccgcaccgc ggcacgatat cgaattccgt ctcggagcac
     1861 tggcgcgcct tcgaatcgat atgtcgagtt tactccctat cagtgataga gaacgtatgt
     1921 cgagtttact ccctatcagt gatagagaac gatgtcgagt ttactcccta tcagtgatag
     1981 agaacgtatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc
     2041 tatcagtgat agagaacgta tgtcgagttt atccctatca gtgatagaga acgtatgtcg
     2101 agtttactcc ctatcagtga tagagaacgt atgtcgaggt aggcgtgtac ggtgggaggc
     2161 ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacata tggccgccac
     2221 catggactac aaagaccatg acggtgatta taaagatcat gatatcgatt acaaggatga
     2281 cgatgacaag acaggtagta caggtagtat gcctgcccct ttggagacct gcatctctga
     2341 tctcgactgc tccagcagca acagcagcag cgacctgtcc agcttcctca ccgacgagga
     2401 ggactgtgcc aggctacagc ccctagcctc cacctcgggg ctgtccgtgc cagcccggag
     2461 gagcgctccc gccctctccg gggcatcgaa tgttcccggt gcccaggacg aagagcagga
     2521 acggcggagg cggcgaggtc gcgctcgggt gcggtccgag gctctgctgc actccctgcg
     2581 gaggagtcgt cgcgtcaaag ccaacgatcg cgagcgcaac cgcatgcaca acctcaacgc
     2641 tgcgctggac gccttgcgca gcgtgctgcc ctcgttcccc gacgacacca agctcaccaa
     2701 gattgagacg ctgcgcttcg cctacaacta catctgggcc ctggctgaga cactgcgcct
     2761 ggcagatcaa gggctccccg ggggcagtgc ccgggagcgc ctcctgcctc cgcagtgtgt
     2821 cccctgtctg cccgggcccc cgagcccggc cagcgacact gagtcctggg gttccggggc
     2881 cgctgcctcc ccctgcgcca ctgtggcatc accactctct gaccccagta gtccctcggc
     2941 ttcagaagac ttcacctatg gcccgggcga tccccttttc tcctttcctg gcctgcccaa
     3001 agacctgctc cacacgacgc cctgtttcat cccataccac tagcaattgt tgttgttaac
     3061 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3121 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     3181 catgtctgcg gatccgatat cagaagccat agagcccacc gcatccccag catgcctgct
     3241 attgtcttcc caatcctccc ccttgctgtc ctgccccacc ccacccccca gaatagaatg
     3301 acacctactc agacaatgcg atgcaatttc ctcattttat taggaaagga cagtgggagt
     3361 ggcaccttcc agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact
     3421 agaaggcaca gtcgaggctg atcagcgagc tctagatcat cgatgcatgg ggtcgtgcgc
     3481 tcctttcggt cgggcgctgc gggtcgtggg gcgggcgtca ggcaccgggc ttgcgggtca
     3541 tgcaccaggt gcgcggtcct tcgggcacct cgacgtcggc ggtgacggtg aagccgagcc
     3601 gctcgtagaa ggggaggttg cggggcgcgg aggtctccag gaaggcgggc accccggcgc
     3661 gctcggccgc ctccactccg gggagcacga cggcgctgcc cagacccttg ccctggtggt
     3721 cgggcgagac gccgacggtg gccaggaacc acgcgggctc cttgggccgg tgcggcgcca
     3781 ggaggccttc catctgttgc tgcgcggcca gccgggaacc gctcaactcg gccatgcgcg
     3841 ggccgatctc ggcgaacacc gcccccgctt cgacgctctc cggcgtggtc cagaccgcca
     3901 ccgcggcgcc gtcgtccgcg acccacacct tgccgatgtc gagcccgacg cgcgtgagga
     3961 agagttcttg cagctcggtg acccgctcga tgtggcggtc cggatcgacg gtgtggcgcg
     4021 tggcggggta gtcggcgaac gcggcggcga gggtgcgtac ggccctgggg acgtcgtcgc
     4081 gggtggcgag gcgcaccgtg ggcttgtact cggtcatggt ggcggcgtgg tggagtacgc
     4141 gcccggggag cccaagggca cgccctggca cccgcaccgc ggagatctcc cgatccgtcg
     4201 ccagcctgct tttttgtaca aagttggcat tataaaaaag cattgcttat caatttgttg
     4261 caacgaacag gtcactatca gtcaaaataa aatcattatt tggggcccga gcttaagtaa
     4321 ctaactaaca ggaagagttt gtagaaacgc aaaaaggcca tccgtcagga tggccttctg
     4381 cttagtttga tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg
     4441 cttcacaacg ttcaaatccg ctcccggcgg atttgtccta ctcaggagag cgttcaccga
     4501 caaacaacag ataaaacgaa aggcccagtc ttccgactga gcctttcgtt ttatttgatg
     4561 cctggcagtt ccctactctc gcttagtagt tagacgtccc cgagatccat gctagcggta
     4621 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtg
//
