LOCUS       sequence-432464-        6240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP and mCD4 transmembrane domain under control of the 
            2C-specific MERVL promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6240)
  AUTHORS   Xie SQ, Leeke BJ, Whilding C, Wagner RT, Garcia-Llagostera F, Low Y,
            Chammas P, Cheung NT, Dormann D, McManus MT, Percharde M
  TITLE     Nucleolar-based Dux repression is essential for embryonic two-cell 
            stage exit.
  JOURNAL   Genes Dev. 2022 Mar 1;36(5-6):331-347. doi: 10.1101/gad.349172.121. 
            Epub 2022 Mar 10.
  PUBMED    35273077
REFERENCE   2  (bases 1 to 6240)
  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: "Genes 
            Dev."; date: "2022-03-1"; volume: "36(5-6):331-347. doi"; pages: " 
            10.1101/gad.349172.121. Epub 2022 Mar 10"
COMMENT     Sequence Label: 2C-EGFP-t2a-mCD4
FEATURES             Location/Qualifiers
     source          1..6240
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      260..848
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     749..768
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     regulatory      1791..1800
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1797..1799,1800..1802,1803..2513)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1797..1799,1800..1802,1803..2513)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1842..1863)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2103..2122)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2450..2471
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2514..2567
                     /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"
     polyA_signal    3990..4111
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4027..4046)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4081..4100
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4118..4573)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4255..4276)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4467..4486
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4600..4704
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4706..5063
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4706..4897
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4729..4749)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4914..5049
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5098..5892
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5098..5892
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5152..5171)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5762..5781
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6080..6099)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6124..6171
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
       61 tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt
      121 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
      181 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
      241 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
      301 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
      361 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
      421 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
      481 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
      541 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
      601 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
      661 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
      721 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
      781 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
      841 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
      901 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc
      961 attagttatt atggtcgacc tgcaggcggc cgcgaattca ctagtgattg cggccgctgt
     1021 agtggttatt cctggttgtc aacttgacaa tatttggaat gaactacaat tcggaattgg
     1081 aaggctcacc agtgaccctt atctggaggc ttggagatcc ttatctggat cttggtttga
     1141 agatcttgag ccatagtggc tatggattcc agaagattga atctccgagt taaggaacac
     1201 acctttaatc tgggctatgc ctttcatctg ggattaaagg tgtggtggaa cacaccttta
     1261 atctgggcta caccttttgc tggagacaat ataaggacat tggaagaagg gagtctagct
     1321 cttgctcctg ctccttcgcc tgcttgctgc gtgagactga gtaactacta gatccttgga
     1381 cttccattca cagctgtgac tgaacgattg ttgggaattg agctgccgac tgtaagtcat
     1441 caataaattc ctttattatc tagagactat tcataagttc tgtgactcta gagaaccctg
     1501 actaatacag aagttggtac caggagtggt tctagagtaa cagaagtaca aggatgaatc
     1561 ttttaaaata ctggaattgg cttgttgatc caccagcact ttcaactatt gaaacctctc
     1621 cagattctct ccctcctggg agctcagaga attttgaaga cccatggttg aaactctatt
     1681 ctgaacttaa agaagctaat gaccttgatt ttcttaatga attaggtgat tcagtgcaca
     1741 aagcttgtcg acaatcgaat tcccgcgggc ccgggatcca ccggccggtc gccaccatgg
     1801 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1861 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1921 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1981 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2041 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2101 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2161 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2221 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2281 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2341 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2401 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2461 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aaggagggca
     2521 gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggcccaccg gacatgtgcc
     2581 gagccatctc tcttaggcgc ttgctgctgc tgctgctgca gctgtcacaa ctcctagctg
     2641 tcactcaagg gaagacgctg gtgctgggga aggaagggga atcagcagaa ctgccctgcg
     2701 agagttccca gaagaagatc acagtcttca cctggaagtt ctctgaccag aggaagattc
     2761 tggggcagca tggcaaaggt gtattaatta gaggaggttc gccttcgcag tttgatcgtt
     2821 ttgattccaa aaaaggggca tgggagaaag gatcgtttcc tctcatcatc aataaactta
     2881 agatggaaga ctctcagact tatatctgtg agctggagaa caggaaagag gaggtggagt
     2941 tgtgggtgtt caaagtgacc ttcagtccgg gtaccagcct gttgcaaggg cagagcctga
     3001 ccctgacctt ggatagcaac tctaaggtct ctaacccctt gacagagtgc aaacacaaaa
     3061 agggtaaagt tgtcagtggt tccaaagttc tctccatgtc caacctaagg gttcaggaca
     3121 gcgacttctg gaactgcacc gtgaccctgg accagaaaaa gaactggttc ggcatgacac
     3181 tctcagtgct gggttttcag agcacagcta tcacggccta taagagtgag ggagagtcag
     3241 cggagttctc cttcccactc aactttgcag aggaaaacgg gtggggagag ctgatgtgga
     3301 aggcagagaa ggattctttc ttccagccct ggatctcctt ctccataaag aacaaagagg
     3361 tgtccgtaca aaagtccacc aaagacctca agctccagct gaaggaaacg ctcccactca
     3421 ccctcaagat accccaggtc tcgcttcagt ttgctggttc tggcaacctg actctgactc
     3481 tggacaaagg gacactgcat caggaagtga acctggtggt gatgaaagtg gctcagctca
     3541 acaatacttt gacctgtgag gtgatgggac ctacctctcc caagatgaga ctgaccctga
     3601 agcaggagaa ccaggaggcc agggtctctg aggagcagaa agtagttcaa gtggtggccc
     3661 ctgagacagg gctgtggcag tgtctactga gtgaaggtga taaggtcaag atggactcca
     3721 ggatccaggt tttatccaga ggggtgaacc agacagtgtt cctggcttgc gtgctgggtg
     3781 gctccttcgg ctttctgggt ttccttgggc tctgcatcct ctgctgtgtc aggtgccggc
     3841 accaacagcg ccagtgagta caagtaaagc ggccgcgact ctagatcata atcagccata
     3901 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     3961 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     4021 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4081 gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg
     4141 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     4201 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     4261 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     4321 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     4381 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     4441 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     4501 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     4561 ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4621 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4681 caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc
     4741 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4801 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4861 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     4921 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4981 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     5041 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg
     5101 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     5161 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     5221 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     5281 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     5341 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     5401 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     5461 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     5521 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     5581 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc
     5641 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     5701 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     5761 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     5821 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     5881 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     5941 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     6001 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     6061 accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta
     6121 tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg
     6181 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
//
