LOCUS       sequence-486779-        7273 bp    DNA     circular SYN 19-SEP-2025
DEFINITION  Express EGFP under SFFV promoter. Xho I-SFFVp-AgeI-EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7273)
  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 7273)
  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_SFFVp-EGFP
FEATURES             Location/Qualifiers
     source          1..7273
                     /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        1946..2353
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      2356..2365
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2362..2364,2365..2367,2368..3081)
                     /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(2362..2364,2365..2367,2368..3078)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2407..2428)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2668..2687)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3015..3036
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3124..3712
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3177..3197)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3595..3606)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3799..4032
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4104..4225
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4141..4160)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4195..4214
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4265..4400
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4420..4439)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4421..4439)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4449..4466)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4449..4465)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4458..4480)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4607..5062
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4694..4713)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4904..4925
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5088..5192
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5193..5261,5262..6053)
                     /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(5411..5430)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6224..6812
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6713..6732
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6966..6983
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7100..7121
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7136..7141,7142..7159,7160..7166)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7174..7190
                     /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     7179..7201
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7198..7214
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7198..7214
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7233..7253
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7235..7253
                     /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 tagccgtaac gccattttgc aaagcatgga aaaataccaa
     1981 accaagaata gagaagttca gatcaagggc gggtacatga aaatagctaa cgttgggcca
     2041 aacaggatat ctgcggtgag cagtttcggc cccggcccgg ggccaagaac agatggtcac
     2101 cgcagtttcg gccccggccc gaggccaaga acagatggtc cccagatatg gcccaaccct
     2161 cagcagtttc ttaagaccca tcagatgttt ccaggctccc ccaaggacct gaaatgaccc
     2221 tgcgccttat ttgaattaac caatcagcct gcttctcgct tctgttcgcg cgcttctgct
     2281 tcccgagctc tataaaagag ctcacaaccc ctcactcggc gcgccagtcc tccgacagac
     2341 tgagtcgccc gggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg
     2401 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     2461 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     2521 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     2581 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     2641 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     2701 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     2761 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     2821 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     2881 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     2941 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     3001 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     3061 atggacgagc tgtacaagta aagggcgaat tccagcacac tggcggccgt tactagtgtc
     3121 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     3181 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     3241 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     3301 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     3361 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     3421 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     3481 ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt tccatggctg
     3541 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     3601 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     3661 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcctggaatt
     3721 cgagctcggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
     3781 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt
     3841 ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     3901 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     3961 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     4021 aaatctctag cagtagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga
     4081 aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa
     4141 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4201 tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc
     4261 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4321 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     4381 aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac
     4441 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     4501 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     4561 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     4621 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4681 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4741 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4801 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     4861 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4921 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     4981 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5041 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5101 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5161 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5221 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5281 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5341 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5401 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5461 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5521 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5581 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5641 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5701 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     5761 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     5821 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     5881 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     5941 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6001 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6061 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6121 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6181 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6241 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6301 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6361 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6421 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6481 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6541 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6601 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6661 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6721 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6781 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6841 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     6901 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     6961 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     7021 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     7081 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     7141 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     7201 gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa
     7261 agctggagct gca
//
