LOCUS       sequence-384989-        6612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SOX17 donor plasmid to generate locus specific C-terminal 
            integration of H2B-mCitrine into the human SOX17-locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6612)
  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 6612)
  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_T2A-H2B-mCitrine_loxP-hPGK-BSD-loxP
FEATURES             Location/Qualifiers
     source          1..6612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     72..94
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(132..150)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        218..322
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(323..391,392..1183)
                     /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(541..560)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1354..1942
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1843..1862
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             2710..2727
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     CDS             2716..2733
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     CDS             3019..3072
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3073..3450
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             join(3469..3471,3472..4185)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3511..3532)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3772..3791)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4119..4140
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4197..4230)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4235..4745
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4664..4682
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4758..5156
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5166..5627
                     /label=PGK poly(A) signal
                     /note="mouse phosphoglycerate kinase 1 polyadenylation 
                     signal"
     protein_bind    complement(5638..5671)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 tttttttgct tttagattta atacatatac ggacgattgc acagacttag gggagaatcc
       61 atgaatggtt tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
      121 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
      181 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
      241 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
      301 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
      361 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
      421 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
      481 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
      541 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
      601 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
      661 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
      721 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
      781 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
      841 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
      901 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
      961 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     1021 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     1081 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     1141 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     1201 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     1261 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     1321 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     1381 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     1441 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     1501 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     1561 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     1621 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     1681 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     1741 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     1801 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     1861 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     1921 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     1981 ttgctggcct tttgctcaca tgtgggcctg gagcgggagc gcagccgata agccctgcgc
     2041 ccctctcccc cttccttcca ctgtgcaggc aagtcgtgga aggcgctgac gctggcggag
     2101 aagcggccct tcgtggagga ggcagagcgg ctgcgcgtgc agcacatgca ggaccacccc
     2161 aactacaagt accggccgcg gcggcgcaag caggtgaagc ggctgaagcg ggtggagggc
     2221 ggcttcctgc acggcctggc tgagccgcag gcggccgcgc tgggccccga gggcggccgc
     2281 gtggccatgg acggcctggg cctccagttc cccgagcagg gcttccccgc cggcccgccg
     2341 ctgctgcctc cgcacatggg cggccactac cgcgactgcc agagtctggg cgcgcctccg
     2401 ctcgacggct acccgttgcc cacgcccgac acgtccccgc tggacggcgt ggaccccgac
     2461 ccggctttct tcgccgcccc gatgcccggg gactgcccgg cggccggcac ctacagctac
     2521 gcgcaggtct cggactacgc tggccccccg gagcctcccg ccggtcccat gcacccccga
     2581 ctcggcccag agcccgcggg tccctcgatt ccgggcctcc tggcgccacc cagcgccctt
     2641 cacgtgtact acggcgcgat gggctcgccc ggggcgggcg gcgggcgcgg cttccagatg
     2701 cagccgcaac accagcacca gcaccagcac cagcaccacc ccccgggccc cggacagccg
     2761 tcgccccctc cggaggcact gccctgccgg gacggcacgg accccagtca gcccgccgag
     2821 ctcctcgggg aggtggaccg cacggaattt gaacagtatc tgcacttcgt gtgcaagcct
     2881 gagatgggcc tcccctacca ggggcatgac tccggtgtga atctccccga cagccacggg
     2941 gccatttcct cggtggtgtc cgacgccagc tccgcggtat attactgcaa ctatcctgac
     3001 gtggaattcg gcagtggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag
     3061 aatcctggcc caatgccaga gccagcgaag tctgctcccg ccccgaaaaa gggctccaag
     3121 aaggcggtga ctaaggcgca gaagaaaggc ggcaagaagc gcaagcgcag ccgcaaggag
     3181 agctattcca tctatgtgta caaggttctg aagcaggtcc accctgacac cggcatttcg
     3241 tccaaggcca tgggcatcat gaattcgttt gtgaacgaca ttttcgagcg catcgcaggt
     3301 gaggcttccc gcctggcgca ttacaacaag cgctcgacca tcacctccag ggagatccag
     3361 acggccgtgc gcctgctgct gcctggggag ttggccaagc acgccgtgtc cgagggtact
     3421 aaggccatca ccaagtacac cagcgctaag gatccaccgg tcgccaccgt gagcaagggc
     3481 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     3541 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     3601 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc
     3661 ggctacggcc tgatgtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     3721 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     3781 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     3841 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     3901 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     3961 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     4021 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag
     4081 tccaagctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     4141 accgccgccg ggatcactct cggcatggac gagctgtaca agtagatagt acttaaataa
     4201 cttcgtatag catacattat acgaagttat aagcggggtt ggggttgcgc cttttccaag
     4261 gcagccctgg gtttgcgcag ggacgcggct gctctgggcg tggttccggg aaacgcagcg
     4321 gcgccgaccc tgggtctcgc acattcttca cgtccgttcg cagcgtcacc cggatcttcg
     4381 ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt
     4441 ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc actagtaccc
     4501 tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca
     4561 atagcggctg ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg
     4621 cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca
     4681 agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc
     4741 cccagcctgc agccaatatg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa
     4801 gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag
     4861 ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac
     4921 cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt
     4981 gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgtgga cggtgccgac
     5041 aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc
     5101 cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaagggg
     5161 atccgctgta agtctgcaga aattgatgat ctattaaaca ataaagatgt ccactaaaat
     5221 ggaagttttt cctgtcatac tttgttaaga agggtgagaa cagagtacct acattttgaa
     5281 tggaaggatt ggagctacgg gggtgggggt ggggtgggat tagataaatg cctgctcttt
     5341 actgaaggct ctttactatt gctttatgat aatgtttcat agttggatat cataatttaa
     5401 acaagcaaaa ccaaattaag ggccagctca ttcctcccac tcatgatcta tagatctata
     5461 gatctctcgt gggatcattg tttttctctt gattcccact ttgtggttct aagtactgtg
     5521 gtttccaaat gtgtcagttt catagcctga agaacgagat cagcagcctc tgttccacat
     5581 acacttcatt ctcagtattg ttttgccaag ttctaattcc atcagaaggt agaattcata
     5641 acttcgtata gcatacatta tacgaagtta tggcgcgcca gccaggtccc tgatccgccc
     5701 cagcctgcag gccagaagca gtgttacaca cttcctggag gagctaagga aatcctcaga
     5761 ctcctgggtt tttgttgttg ctgttgttgt tttttaaaag gtgtgttggc atataattta
     5821 tggtaattta ttttgtctgc cacttgaaca gtttgggggg gtgaggtttc atttaaaatt
     5881 tgttcagaga tttgtttccc atagttggat tgtcaaaacc ctatttccaa gttcaagtta
     5941 actagctttg aatgtgtccc aaaacagctt cctccatttc ctgaaagttt attgatcaaa
     6001 gaaatgttgt cctgggtgtg ttttttcaat cttctaaaaa ataaaatctg gaatcctgct
     6061 tttttgctct actagtacct ctgtcacact agtcttatca aaaaccagtt cttaagatca
     6121 atgttaagtt tattagttaa tgtaaatttc tcatcctcga aaagggtgaa cataaatgcc
     6181 tttaaggagt atatctaaaa ataaacatta ggatatctaa gtttgatgta attgtttcag
     6241 gaaggaaaaa agaaaagcat tctggaatga gcctacttca agtaatctta gtttctaaaa
     6301 ctaacagtta atattttcaa ttccagtata tcactttaag tagaagggga tgtccaagta
     6361 attttggttt tctaactgtt gaatcataag cttgacctgc ccccagaggc tttttggatg
     6421 tttttatctg tgttttgcca tctctttaca ctcctcgaca ttcagtttac cttaatcttc
     6481 acatttttac accttgggaa gtggcaagca tcgctgggtt taagataaag gagtcacaaa
     6541 aactaatcaa aataaaattt gcattatgac aacttttaat acactttagc attttatttg
     6601 attggttttt tt
//
