LOCUS       sequence-486776-        7955 bp    DNA     circular SYN 19-SEP-2025
DEFINITION  Express EGFP under miniUCOE-SFFV promoter. Xho 
            I-miniUCOE-SFFVp-AgeI-EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7955)
  AUTHORS   Kwak CS, Oflaz FE, Qiu J, Wang X
  TITLE     Human stem cell-specific epigenetic signatures control transgene 
            expression.
  JOURNAL   Biochim Biophys Acta Gene Regul Mech. 2024 Dec;1867(4):195063. doi: 
            10.1016/j.bbagrm.2024.195063. Epub 2024 Oct 20.
  PUBMED    39437851
REFERENCE   2  (bases 1 to 7955)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 19, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Biochim 
            Biophys Acta Gene Regul Mech."; date: "2024-12"; volume: 
            "1867(4):195063. doi"; pages: " 10.1016/j.bbagrm.2024.195063. Epub 
            2024 Oct 20"
COMMENT     Sequence Label: pRRL_miniUCOE-SFFVp-EGFP
FEATURES             Location/Qualifiers
     source          1..7955
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        8..234
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             235..415
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    462..587
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1080..1313
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1498..1542
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1809..1926
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2628..3035
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      3038..3047
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3044..3046,3047..3049,3050..3763)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3044..3046,3047..3049,3050..3760)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3089..3110)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3350..3369)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3697..3718
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3806..4394
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3859..3879)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4277..4288)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4481..4714
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4786..4907
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4823..4842)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4877..4896
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4947..5082
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5102..5121)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5103..5121)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5131..5148)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5131..5147)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5140..5162)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5289..5744
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5376..5395)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5586..5607
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5770..5874
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5875..5943,5944..6735)
                     /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(6093..6112)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6906..7494
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7395..7414
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7648..7665
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7782..7803
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7818..7823,7824..7841,7842..7848)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7856..7872
                     /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     7861..7883
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7880..7896
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7880..7896
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7915..7935
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7917..7935
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag
       61 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg
      121 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact
      181 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct
      241 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      301 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      361 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
      421 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
      481 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
      541 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
      601 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
      661 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
      721 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
      781 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
      841 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
      901 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
      961 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1021 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1081 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1141 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1201 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1261 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1321 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1381 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     1441 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     1501 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     1561 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     1621 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     1681 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     1741 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat
     1801 cggttaactt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     1861 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     1921 aattttatcg atcacgagac tagcccggga ggtggtccct gcagttacgt caatgataac
     1981 ccccgccaga aaaatcttag tagccttccc tttttgtttt ccgtgcccca actcggcgga
     2041 ttgactcggc cccttccgga aacacccgaa tcaacttcta gtcaaattat tgttcacgcc
     2101 gcaatgaccc acccctggcc cgcgtctgtg gaactgaccc ctggtgtaca ggagagttcg
     2161 ctgctgaaag tggtcccaaa ggggtactag tttttaagct cccaactccc cctcccccag
     2221 cgtctggagg attccacacc ctcgcaccgc aggggcgagg aagtgggcgg agtccggttt
     2281 tggcgccagc cgctgaggct gccaagcaga aaagccaccg ctgaggagac tccggtcact
     2341 gtcctcgccc cgcctccccc ttccctcccc ttggggacca ccgggcgcca cgccgcgaac
     2401 ggtaagtgcc gcggtcgtcg gcgcctccgc cctcccccta gggccccaat tcccagcggg
     2461 cgcggcgcgc ggccccgcgc gcagctcccg gctccctccc ccttcggatg tggcttgagc
     2521 tgtaggcgcg gagtcctgca gccccgataa aataaaagat tttatttagt ctccagaaaa
     2581 aggggggaat gaaagacccc acctgtaggt ttggcaagct agctgcagta acgccatttt
     2641 gcaaggcatg gaaaaatacc aaaccaagaa tagagaagtt cagatcaagg gcgggtacat
     2701 gaaaatagct aacgttgggc caaacaggat atctgcggtg agcagtttcg gccccggccc
     2761 ggggccaaga acagatggtc accgcagttt cggccccggc ccgaggccaa gaacagatgg
     2821 tccccagata tggcccaacc ctcagcagtt tcttaagacc catcagatgt ttccaggctc
     2881 ccccaaggac ctgaaatgac cctgcgcctt atttgaatta accaatcagc ctgcttctcg
     2941 cttctgttcg cgcgcttctg cttcccgagc tctataaaag agctcacaac ccctcactcg
     3001 gcgcgccagt cctccgacag actgagtcgc ccgggtcgcc accatggtga gcaagggcga
     3061 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     3121 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     3181 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     3241 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     3301 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     3361 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     3421 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     3481 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     3541 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     3601 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     3661 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     3721 cgccgccggg atcactctcg gcatggacga gctgtacaag taaagggcga attccagcac
     3781 actggcggcc gttactagtg tcgacaatca acctctggat tacaaaattt gtgaaagatt
     3841 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     3901 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     3961 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     4021 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     4081 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     4141 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     4201 gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     4261 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     4321 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     4381 ggccgcctcc ccgcctggaa ttcgagctcg gtacctttaa gaccaatgac ttacaaggca
     4441 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     4501 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
     4561 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     4621 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     4681 agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt
     4741 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca
     4801 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4861 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc
     4921 tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     4981 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     5041 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc
     5101 gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg
     5161 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     5221 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     5281 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     5341 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     5401 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     5461 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     5521 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     5581 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     5641 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     5701 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     5761 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5821 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5881 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5941 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6001 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6061 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6121 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6181 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6241 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6301 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6361 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6421 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6481 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6541 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6601 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6661 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6721 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6781 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6841 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6901 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6961 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     7021 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     7081 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7141 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7201 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7261 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7321 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7381 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7441 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7501 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7561 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7621 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     7681 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     7741 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     7801 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     7861 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
     7921 aaccctcact aaagggaaca aaagctggag ctgca
//
