LOCUS       Exported                8069 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    EF.PGK.GFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8069)
  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 8069)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8069
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    55..76
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        91..121
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    129..145
                     /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     134..156
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     153..169
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     153..169
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     188..208
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        190..208
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        236..462
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             463..643
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    690..815
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1308..1541
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1726..1770
                     /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        2158..3336
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2389..3327
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3284..3304
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        3353..3371
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3453..3963
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3882..3900
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      3979..3988
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             3985..4704
                     /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             3985..4701
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4030..4051)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4291..4310)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4638..4659
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4823..5056
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5128..5249
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5165..5184)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5219..5238
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5289..5424
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5351..5370
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        complement(5445..5463)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5473..5490)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5473..5489)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5482..5504)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5631..6086
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5718..5737)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5928..5949
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6112..6216
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6217..7077
                     /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(6435..6454)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7248..7836
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7737..7756
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7990..8007
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
       61 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      121 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      181 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag
      241 tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta
      301 caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc
      361 ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat
      421 tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac
      481 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
      541 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
      601 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg
      661 acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag
      721 cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg
      781 gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat
      841 cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata
      901 tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat
      961 cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag
     1021 aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga
     1081 taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca
     1141 ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg
     1201 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
     1261 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
     1321 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
     1381 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
     1441 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
     1501 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
     1561 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg
     1621 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
     1681 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
     1741 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
     1801 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
     1861 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
     1921 cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag
     1981 agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg atcacgagac
     2041 tagcctcgag aagcttgatg acggtatcga taagctttgc aaagatggat aaagttttaa
     2101 acagagagga atctttgcag ctaatggacc ttctaggtct tgaaaggagt gggaattggc
     2161 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     2221 ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     2281 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     2341 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg
     2401 tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact
     2461 tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag tgggtgggag
     2521 agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga ggcctggcct
     2581 gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc
     2641 gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa
     2701 gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt tggggccgcg
     2761 ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg
     2821 cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct ggtgcctggc
     2881 ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt
     2941 gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa atggaggacg
     3001 cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc
     3061 tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca cctcgattag
     3121 ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta tgcgatggag
     3181 tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt gatgtaattc
     3241 tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc tcagacagtg
     3301 gttcaaagtt tttttcttcc atttcaggtg tcgtgaggaa ttagcttggt actaatacga
     3361 ctcactatag ggagacccaa gctggctagg taagcttgat atcgaattcc tgcagcccgg
     3421 gggatctcga gaagcttgat atcgaattcc acggggttgg ggttgcgcct tttccaaggc
     3481 agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa acgcagcggc
     3541 gccgaccctg ggtctcgcac attcttcacg tccgttcgca gcgtcacccg gatcttcgcc
     3601 gctacccttg tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc
     3661 ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg cacgtctcac tagtaccctc
     3721 gcagacggac agcgccaggg agcaatggca gcgcgccgac cgcgatgggc tgtggccaat
     3781 agcggctgct cagcagggcg cgccgagagc agcggccggg aaggggcggt gcgggaggcg
     3841 gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag
     3901 cctccggagc gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga cctctctccc
     3961 cagggggatc caccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg
     4021 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     4081 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     4141 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     4201 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     4261 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     4321 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     4381 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     4441 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     4501 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     4561 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     4621 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     4681 ggcatggacg agctgtacaa gtaaagcggc cgcgtcgacg ggccctcgac gggcccgcgg
     4741 aattcgagct cggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac
     4801 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg
     4861 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     4921 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     4981 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     5041 tggaaaatct ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca
     5101 aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa
     5161 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     5221 ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa
     5281 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     5341 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     5401 agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta
     5461 ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     5521 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     5581 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
     5641 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     5701 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5761 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5821 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     5881 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5941 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     6001 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     6061 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     6121 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     6181 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     6241 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     6301 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     6361 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     6421 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     6481 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     6541 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6601 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6661 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6721 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6781 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6841 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6901 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6961 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     7021 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     7081 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     7141 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     7201 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     7261 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     7321 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     7381 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     7441 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     7501 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     7561 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     7621 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7681 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7741 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7801 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7861 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     7921 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     7981 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     8041 ctccccgcgc gttggccgat tcattaatg
//
