LOCUS       sequence-486777-        7482 bp    DNA     circular SYN 24-SEP-2025
DEFINITION  Express EGFP under CMV promoter. Xho I-CMVp-AgeI-EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7482)
  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 7482)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 24, 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_CMVp-EGFP
FEATURES             Location/Qualifiers
     source          1..7482
                     /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"
     enhancer        1996..2299
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2300..2503
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2454..2474
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2526..2550
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2565..2574
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2571..2573,2574..2576,2577..3290)
                     /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(2571..2573,2574..2576,2577..3287)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2616..2637)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2877..2896)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3224..3245
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3333..3921
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3386..3406)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3804..3815)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4008..4241
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4313..4434
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4350..4369)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4404..4423
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4474..4609
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4629..4648)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4630..4648)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4658..4675)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4658..4674)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4667..4689)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4816..5271
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4903..4922)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5113..5134
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5297..5401
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5402..5470,5471..6262)
                     /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(5620..5639)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6433..7021
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6922..6941
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7175..7192
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7309..7330
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7345..7350,7351..7368,7369..7375)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7383..7399
                     /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     7388..7410
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7407..7423
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7407..7423
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7442..7462
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7444..7462
                     /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 tagccatagt aatcaattac ggggtcatag ttcatagccc
     1981 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     2041 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     2101 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     2161 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     2221 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     2281 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     2341 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     2401 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     2461 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactgtcgg
     2521 gatcaacaag tttgtacaaa aaagcaggct tcgaaggaga tatcgccacc atggtgagca
     2581 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     2641 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2701 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2761 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     2821 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2881 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2941 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     3001 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     3061 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     3121 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     3181 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     3241 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa agggcgaatt
     3301 ccagcacact ggcggccgtt actagtgtcg acaatcaacc tctggattac aaaatttgtg
     3361 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     3421 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     3481 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     3541 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     3601 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     3661 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     3721 cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg
     3781 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     3841 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     3901 ccctttgggc cgcctccccg cctggaattc gagctcggta cctttaagac caatgactta
     3961 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     4021 tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca
     4081 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     4141 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     4201 atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct
     4261 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt
     4321 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4381 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4441 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4501 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4561 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac
     4621 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     4681 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     4741 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     4801 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4861 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4921 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4981 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5041 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5101 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5161 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5221 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     5281 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5341 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5401 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5461 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5521 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5581 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5641 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5701 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5761 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5821 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5881 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5941 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6001 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6061 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6121 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6181 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6241 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6301 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6361 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6421 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6481 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6541 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6601 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6661 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6721 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6781 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6841 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6901 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6961 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7021 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7081 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     7141 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     7201 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     7261 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     7321 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     7381 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     7441 cgcaattaac cctcactaaa gggaacaaaa gctggagctg ca
//
