LOCUS       sequence-384991-        7665 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T-REX17 donor plasmid to generate locus specific integration of 
            mRuby followed by 3xSV40 poly(A) into Exon 1 of human lncRNA 
            T-REX17.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7665)
  AUTHORS   Landshammer A, Bolondi A, Kretzmer H, Much C, Buschow R, Rose A, Wu 
            HJ, Mackowiak SD, Braendl B, Giesselmann P, Tornisiello R, Parsi KM,
            Huey J, Mielke T, Meierhofer D, Maehr R, Hnisz D, Michor F, Rinn JL,
            Meissner A
  TITLE     T-REX17 is a transiently expressed non-coding RNA essential for 
            human endoderm formation.
  JOURNAL   Elife. 2023 Jan 31;12:e83077. doi: 10.7554/eLife.83077.
  PUBMED    36719724
REFERENCE   2  (bases 1 to 7665)
  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: "Elife."; 
            date: "2023-01-31"; volume: "12:e83077. doi"; pages: " 
            10.7554/eLife.83077"
COMMENT     Sequence Label: 
            pUC19_mRuby-3xFLAG-NLS-3xSV40-poly(A)_loxP-PGK-Puro-loxP
FEATURES             Location/Qualifiers
     source          1..7665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    1283..1316
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1795..2394)
                     /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(1878..1898)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2375..2394
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(2486..2505)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     protein_bind    2907..2940
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2960..3081
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3112..3233
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3264..3385
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(3419..3466)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(3467..3532)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(3533..4243)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     regulatory      4240..4249
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(5269..5286)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5269..5285)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5278..5300)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5494..5513)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5613..5635
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5673..5691)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5759..5863
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5864..5932,5933..6724)
                     /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     complement(6082..6101)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6895..7483
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7384..7403
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7637..7654
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc
      241 atgcctgcag gtcgactcta gaggatcccc ttgagcagct ggcctggggt cacccacaaa
      301 aggtgaatgt cagggctggt gttcttttct tccagttaca ctttatgggc tcctgtccag
      361 acataggagt acttgttaaa cactgcaagt acttagacac ctacttgaag gggtcagcct
      421 aagagaccca gcttttctct tctgagtgcc cccacctcag aggctttctt gtcttagggc
      481 atcagtacca agatctgtct gtatcaaggt cctctgaccc aaggaggaag ggaaaggcag
      541 agaacttgat cacagaacag attggcaacc accacatttt ccttcttgga aacatctggg
      601 ttttcgtttc cctgctgctc agtagaaaac acccagctat gtaaaacctc ctagttagaa
      661 gtcaccgcca gctctgtgta ggaggtgaga tgtgaaatag atgatttcca tgtaattttc
      721 tatcatccca gggccttctt agtaactgtc tccaatcctg gttattcaat cctggtgcag
      781 acaatgccaa tacaaagatg tcatgtgacc cagaaatccg tgtgcagcaa gaaaccttgg
      841 tagccataga gtttttgttg ttttactcca gggatttaaa aatccactta gtgattaatg
      901 cccattccag gcttatgtac agcaaaacaa tctcttgcag ttgaagttgg cctttatatt
      961 ccctttagaa tgaaagtatc aaaccatcca ctgatatact taaaagcctt aagagcatct
     1021 tccatgtgta tagagtgtcc ttgtagttcc aaaggtggct ttgcagggag gcagtagctc
     1081 tggcttggca gttccaaaga tagtttttcc tgggccttct ctcttgaaat aggagccata
     1141 ggttttatat atacttaata aaaatcagag aaaagacaag agatttccag tgtccccaaa
     1201 tcagatggag gagcctgtaa accatcctta cttacatggc cagcgtggtg gcagcccagg
     1261 tggtgagagg agctggcgcg ccataacttc gtataatgta tgctatacga agttataggc
     1321 ctctagagtc agcttctgat ggaattagaa cttggcaaaa caatactgag aatgaagtgt
     1381 atgtggaaca gaggctgctg atctcgttct tcaggctatg aaactgacac atttggaaac
     1441 cacagtactt agaaccacaa agtgggaatc aagagaaaaa caatgatccc acgagagatc
     1501 tatagatcta tagatcatga gtgggaggaa tgagctggcc cttaatttgg ttttgcttgt
     1561 ttaaattatg atatccaact atgaaacatt atcataaagc aatagtaaag agccttcagt
     1621 aaagagcagg catttatcta atcccacccc acccccaccc ccgtagctcc aatccttcca
     1681 ttcaaaatgt aggtactctg ttctcaccct tcttaacaaa gtatgacagg aaaaacttcc
     1741 attttagtgg acatctttat tgtttaatag atcatcaatt tctgcagagg atcctcaggc
     1801 accgggcttg cgggtcatgc accaggtgcg cggtccttcg ggcacctcga cgtcggcggt
     1861 gacggtgaag ccgagccgct cgtagaaggg gaggttgcgg ggcgcggagg tctccaggaa
     1921 ggcgggcacc ccggcgcgct cggccgcctc cactccgggg agcacgacgg cgctgcccag
     1981 acccttgccc tggtggtcgg gcgagacgcc gacggtggcc aggaaccacg cgggctcctt
     2041 gggccggtgc ggcgccagga ggccttccat ctgttgctgc gcggccagcc gggaaccgct
     2101 caactcggcc atgcgcgggc cgatctcggc gaacaccgcc cccgcttcga cgctctccgg
     2161 cgtggtccag accgccaccg cggcgccgtc gtccgcgacc cacaccttgc cgatgtcgag
     2221 cccgacgcgc gtgaggaaga gttcttgcag ctcggtgacc cgctcgatgt ggcggtccgg
     2281 gtcgacggtg tggcgcgtgg cggggtagtc ggcgaacgcg gcggcgaggg tgcgtacggc
     2341 ccgggggacg tcgtcgcggg tggcgaggcg caccgtgggc ttgtactcgg tcatgctagc
     2401 taagcttcag ctgctcgaga tctagatgga tgcaggtcga aaggcccgga gatgaggaag
     2461 aggagaacag cgcggcagac gtgcgctttt gaagcgtgca gaatgccggg cctccggagg
     2521 accttcgggc gcccgccccg cccctgagcc cgcccctgag cccgcccccg gacccacccc
     2581 ttcccagcct ctgagcccag aaagcgaagg agcaaagctg ctattggccg ctgccccaaa
     2641 ggcctacccg cttccattgc tcagcggtgc tgtccatctg cacgagacta gtgagacgtg
     2701 ctacttccat ttgtcacgtc ctgcacgacg cgagctgcgg ggcggggggg aacttcctga
     2761 ctaggggagg agtagaaggt ggcgcgaagg ggccaccaaa gaacggagcc ggttggcgcc
     2821 taccggtgga tgtggaatgt gtgcgaggcc agaggccact tgtgtagcgc caagtgccca
     2881 gcggggctgc taaagcgcat gcgcttataa cttcgtataa tgtatgctat acgaagttat
     2941 ttaagtacta ttggagacta acttgtttat tgcagcttat aatggttaca aataaagcaa
     3001 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3061 caaactcatc aatgtatctt atcatgtctg ctcgaagcgg ccgctctaga aaacttgttt
     3121 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     3181 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     3241 tgctcgaagc ggccgctcta gaaaacttgt ttattgcagc ttataatggt tacaaataaa
     3301 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     3361 tgtccaaact catcaatgta tcttatcatg tctgctcgaa gcggccgctc tagaatcatt
     3421 tcttcttctt agcctgtcca gccttctttg tggcggcagg tcgcttctta tcgtcatcgt
     3481 ctttgtaatc aatatcatga tccttgtagt ctccgtcgtg gtccttatag tccttgtaca
     3541 gctcgtccat cccaccacca agcccggcga acttggcaac tgcgtgttcg cgttgtacta
     3601 cgaacatttc attgtcactt tcctctaacc tttccaggcg gtgatcaacg gcatggatac
     3661 cgggcatctt gatgttcccg acggtctttt ttgacctgta agttgttacg aaagagcaag
     3721 acagatggcc accaccatca actttcagtg ccatatgagt gtatcccctc agaccaccat
     3781 ctgctggata catcatctct gtattaggct cccaaccctt ggtcttcttc tgcatcacgg
     3841 gaccattgga gggaaagttt acccctctga cttggacgtg gtaaacgaga cagccatcct
     3901 caaggctggt gtcctgcatg acggtgacga ctccaccatc ttcgtatctc gtaactcttt
     3961 cccaagtaaa accctcagga aaggactgtt taaagaaatc aggaatgcct ttcgggtact
     4021 tgataaaagt acggctgcca tacatgaacg acgtggcaag aatgtcaaag gcaaatggca
     4081 ggggtcctcc ctcgatgact ttgatcctca tggtttgagt tcccatgtac ggattgcctt
     4141 ctccttcacc tgtgcatttg aattggtggc cgttgaccga accttccatg accaccttca
     4201 tacgcatatt ttccttgatc agctcttcgc ccttagacac catggtggca ccatcttcag
     4261 ggggacagac ctggagactg aaaatgaagg gaaaatgtgg aaaactggga tttaaccaac
     4321 tcgcccttgc atctgccaca atgggacatt gttaggcctg ggcttcatga aacaatagac
     4381 ttcgatagga cataataccc attcagaggt cagctttaca tcctagctct tgggccactg
     4441 agctcccttt gaatttgcaa ctgctgcaca caatgtgtgt gttacagatt atttggccta
     4501 agtctcttcc tgttctccct cccccatgac ttaacttctg ctctactctt ccagtaagtc
     4561 caggagggct tttctctctg gaaatgttgg gaatttgcca cagggctctc cactgccgaa
     4621 atccacacca ctgcttttcc tggccatttc aaaatgagat gagtacatcg tatgaatgga
     4681 ttttaagcac acattcgttc aaatgtgtct gtaccccaaa ttctccaaga gtgtaatcca
     4741 tccctatgct aaatcactgg gtgttttagg tgaagaatct actatttatt aatgaaggac
     4801 cacgctcatt ttttatagtg gaatgcattg taaatacaaa ccattgaagc cttcataatt
     4861 ttttcaaaga tataacccac caaagagaaa agtgaaaaag aataaaagca tgctaataaa
     4921 aaggcttgtt gtagcaaaaa cctgaataga atattaatat ccagtgttat acaactttta
     4981 caacgtgatc tatcctagag ccattttgta gtataacatg agagctgtgc tggcaaaaaa
     5041 taaagtcgca taaaaaagta gaatataaaa ctataagaaa ccatttctgc atatggacaa
     5101 atgatgcaaa gtaataacta aaaataaaaa tagttgtaat gtttggggga tttgggagag
     5161 tatggctctt tacagtcttt attttatttc aaattttttt aaattttatt tttatataga
     5221 gacgggattt caccatgttg cccaggctgg ggtaccgagc tcgaattcac tggccgtcgt
     5281 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     5341 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     5401 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     5461 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5521 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5581 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5641 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5701 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5761 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5821 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5881 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5941 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     6001 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     6061 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     6121 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     6181 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6241 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6301 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6361 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6421 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6481 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6541 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6601 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6661 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6721 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6781 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6841 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6901 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6961 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     7021 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     7081 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     7141 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     7201 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7261 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7321 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7381 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7441 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7501 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7561 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7621 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaaga
//
