LOCUS       sequence-261925-        8206 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8206)
  AUTHORS   Yu X, Zhan X, D'Costa J, Tanavde VM, Ye Z, Peng T, Malehorn MT, Yang
            X, Civin CI, Cheng L
  TITLE     Lentiviral vectors with two independent internal promoters transfer 
            high-level expression of multiple transgenes to human hematopoietic 
            stem-progenitor cells.
  JOURNAL   Mol Ther. 2003 Jun . 7(6):827-38.
  PUBMED    12788657
REFERENCE   2  (bases 1 to 8206)
  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: "Mol Ther. 
            2003 Jun . 7(6):827-38."
COMMENT     Sequence Label: EF.CMV.RFP
FEATURES             Location/Qualifiers
     source          1..8206
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        26..130
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(131..199,200..991)
                     /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(349..368)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1162..1750
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1651..1670
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1904..1921
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2038..2059
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2074..2079,2080..2097,2098..2104)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2112..2128
                     /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     2117..2139
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2136..2152
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2136..2152
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2171..2191
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2173..2191
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2219..2445
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             2446..2626
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2673..2798
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3291..3524
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3709..3753
                     /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"
     promoter        4141..5319
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4372..5310
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5267..5287
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        5336..5354
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        5455..5758
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5759..5962
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5913..5933
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      6044..6053
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6050..6727
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(6230..6250)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     6642..6665
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     LTR             6943..7176
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7248..7369
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7285..7304)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7339..7358
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7409..7544
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(7565..7583)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7593..7610)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7593..7609)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7602..7624)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7751..8179
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7838..7857)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8048..8069
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
       61 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
      121 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
      181 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
      241 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
      301 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
      361 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
      421 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
      481 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
      541 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
      601 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
      661 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
      721 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
      781 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
      841 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
      901 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
      961 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     1021 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     1081 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     1141 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     1201 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     1261 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     1321 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     1381 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     1441 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     1501 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     1561 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     1621 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     1681 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     1741 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     1801 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     1861 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     1921 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     1981 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     2041 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     2101 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     2161 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg
     2221 tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc
     2281 ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt
     2341 gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg
     2401 cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta
     2461 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     2521 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     2581 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     2641 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     2701 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     2761 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     2821 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     2881 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     2941 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     3001 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     3061 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     3121 ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat
     3181 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     3241 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     3301 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     3361 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     3421 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     3481 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     3541 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     3601 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     3661 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     3721 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     3781 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     3841 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     3901 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     3961 gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta tcgatcacga
     4021 gactagcctc gagaagcttg atgacggtat cgataagctt tgcaaagatg gataaagttt
     4081 taaacagaga ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt
     4141 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
     4201 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
     4261 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
     4321 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
     4381 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
     4441 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
     4501 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
     4561 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
     4621 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
     4681 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
     4741 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
     4801 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
     4861 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     4921 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     4981 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     5041 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     5101 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     5161 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     5221 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     5281 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gaattagctt ggtactaata
     5341 cgactcacta tagggagacc caagctggct aggtaagctt gatatcgaat tcctgcagcc
     5401 ctaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     5461 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     5521 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     5581 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     5641 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     5701 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     5761 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     5821 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     5881 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     5941 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggac
     6001 tcagatctcg agctcaagct tcgaattggg ggatccaccg gtcgccacca tggcctcctc
     6061 cgagaacgtc atcaccgagt tcatgcgctt caaggtgcgc atggagggca ccgtgaacgg
     6121 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggcc acaacaccgt
     6181 gaagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccccca
     6241 gttccagtac ggctccaagg tgtacgtgaa gcaccccgcc gacatccccg actacaagaa
     6301 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
     6361 ggcgaccgtg acccaggact cctccctgca ggacggctgc ttcatctaca aggtgaagtt
     6421 catcggcgtg aacttcccct ccgacggccc cgtgatgcag aagaagacca tgggctggga
     6481 ggcctccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga cccacaaggc
     6541 cctgaagctg aaggacggcg gccactacct ggtggagttc aagtccatct acatggccaa
     6601 gaagcccgtg cagctgcccg gctactacta cgtggacgcc aagctggaca tcacctccca
     6661 caacgaggac tacaccatcg tggagcagta cgagcgcacc gagggccgcc accacctgtt
     6721 cctgtagcgg ccgcgactct agatcataat cagccatacc acatttgtag aggttttact
     6781 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     6841 tgttgttgac gggcccgcgg aattcgagct cggtaccttt aagaccaatg acttacaagg
     6901 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     6961 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct
     7021 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     7081 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     7141 tcagaccctt ttagtcagtg tggaaaatct ctagcagtag tagttcatgt catcttatta
     7201 ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg
     7261 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     7321 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc
     7381 tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     7441 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     7501 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat
     7561 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac
     7621 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     7681 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     7741 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     7801 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     7861 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     7921 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     7981 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     8041 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     8101 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     8161 caaaaattta acgcgaattt taacaaaata ttaacgctta caattt
//
