LOCUS       sequence-180614-        7844 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of H2BGFP fusion using PGK promoter in a 
            lentiviral backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7844)
  TITLE     Torok-Storb lab lentiviral fluorescent protein
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7844)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pRRlsinPGK_H2BGFP_WPRE
FEATURES             Location/Qualifiers
     source          1..7844
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     165..184
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     424..445
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      487..496
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(509..886)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     promoter        complement(913..1423)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(976..994)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    1469..1586
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(1853..1897)
                     /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    2082..2315
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    2808..2933
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             2980..3160
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(3161..3387)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(3415..3435)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3415..3433)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3454..3470)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3454..3470)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3467..3489)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3478..3494
                     /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)."
     promoter        complement(join(3502..3508,3509..3526,3527..3532))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3685..3702)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3856..4444)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3936..3955)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4615..5406,5407..5475))
                     /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     5238..5257
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5476..5580)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5606..6061)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5743..5764)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5955..5974
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6188..6210
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6202..6219
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6203..6219
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6229..6248
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6229..6247
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      6268..6403
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6443..6564
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6454..6473)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6508..6527
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             6636..6869
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    6956..7544
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             7062..7073
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7471..7491
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(join(7615..7844,1..484,485..487,488..490))
                     /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             complement(join(7618..7844,1..484,485..487,488..490))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7660..7681)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt
       61 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
      121 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt
      181 cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga
      241 agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg
      301 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg
      361 tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt
      421 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
      481 tgctcaccat ggtggcgacc ggtggatcct tagcgctggt gtacttggtg atggccttag
      541 taccctcgga cacggcgtgc ttggccaact ccccaggcag cagcaggcgc acggccgtct
      601 ggatctccct ggaggtgatg gtcgagcgct tgttgtaatg cgccaggcgg gaagcctcac
      661 ctgcgatgcg ctcgaaaatg tcgttcacaa acgaattcat gatgcccatg gccttggacg
      721 aaatgccggt gtcagggtgg acctgcttca gaaccttgta cacatagatg gaatagctct
      781 ccttgcggct gcgcttgcgc ttcttgccgc ctttcttctg cgccttagtc accgccttct
      841 tggagccctt tttcggggcg ggagcagact tcgctggctc tggcatggtg gcggtaccgt
      901 cgactgcagc ccctggggag agaggtcggt gattcggtca acgagggagc cgactgccga
      961 cgtgcgctcc ggaggcttgc agaatgcgga acaccgcgcg ggcaggaaca gggcccacac
     1021 taccgcccca caccccgcct cccgcaccgc cccttcccgg ccgctgctct cggcgcgccc
     1081 tgctgagcag ccgctattgg ccacagccca tcgcggtcgg cgcgctgcca ttgctccctg
     1141 gcgctgtccg tctgcgaggg tactagtgag acgtgcggct tccgtttgtc acgtccggca
     1201 cgccgcgaac cgcaaggaac cttcccgact taggggcgga gcaggaagcg tcgccggggg
     1261 gcccacaagg gtagcggcga agatccgggt gacgctgcga acggacgtga agaatgtgcg
     1321 agacccaggg tcggcgccgc tgcgtttccc ggaaccacgc ccagagcagc cgcgtccctg
     1381 cgcaaaccca gggctgcctt ggaaaaggcg caaccccaac cccgtggaat tcgatatcaa
     1441 gcttctcgag gctagtctcg tgatcgataa aattttgaat ttttgtaatt tgtttttgta
     1501 attctttagt ttgtatgtct gttgctatta tgtctactat tctttcccct gcactgtacc
     1561 ccccaatccc cccttttctt ttaaaagtta accgataccg tcgagatccg ttcactaatc
     1621 gaatggatct gtctctgtct ctctctccac cttcttcttc tattccttcg ggcctgtcgg
     1681 gtcccctcgg ggttgggagg tgggtctgaa acgataatgg tgaatatccc tgcctaactc
     1741 tattcactat agaaagtaca gcaaaaacta ttcttaaacc taccaagcct cctactatca
     1801 ttatgaataa ttttatatac cacagccaat ttgttatgtt aaaccaattc cacaaacttg
     1861 cccatttatc taattccaat aattcttgtt cattcttttc ttgctggttt tgcgattctt
     1921 caattaagga gtgtattaag cttgtgtaat tgttaatttc tctgtcccac tccatccagg
     1981 tcgtgtgatt ccaaatctgt tccagagatt tattactcca actagcattc caaggcacag
     2041 cagtggtgca aatgagtttt ccagagcaac cccaaatccc caggagctgt tgatccttta
     2101 ggtatctttc cacagccagg attcttgcct ggagctgctt gatgccccag actgtgagtt
     2161 gcaacagatg ctgttgcgcc tcaatagccc tcagcaaatt gttctgctgc tgcactatac
     2221 cagacaataa ttgtctggcc tgtaccgtca gcgtcattga cgctgcgccc atagtgcttc
     2281 ctgctgctcc caagaaccca aggaacaaag ctcctattcc cactgctctt ttttctctct
     2341 gcaccactct tctctttgcc ttggtgggtg ctactcctaa tggttcaatt tttactactt
     2401 tatatttata taattcactt ctccaattgt ccctcatatc tcctcctcca ggtctgaaga
     2461 tcagcggccg cttgctgtgc ggtggtctta cttttgtttt gctcttcctc tatcttgtct
     2521 aaagcttcct tggtgtcttt tatctctatc ctttgatgca cacaatagag ggttgctact
     2581 gtattatata atgatctaag ttcttctgat cctgtctgaa gggatggttg tagctgtccc
     2641 agtatttgtc tacagccttc tgatgtttct aacaggccag gattaactgc gaatcgttct
     2701 agctccctgc ttgcccatac tatatgtttt aatttatatt ttttctttcc ccctggcctt
     2761 aaccgaattt tttcccatcg cgatctaatt ctcccccgct taatactgac gctctcgcac
     2821 ccatctctct ccttctagcc tccgctagtc aaaatttttg gcgtactcac cagtcgccgc
     2881 ccctcgcctc ttgccgtgcg cgcttcagca agccgagtcc tgcgtcgaga gagctcctct
     2941 ggtttccctt tcgctttcaa gtccctgttc gggcgccact gctagagatt ttccacactg
     3001 actaaaaggg tctgagggat ctctagttac cagagtcaca caacagacgg gcacacacta
     3061 cttgaagcac tcaaggcaag ctttattgag gcttaagcag tgggttccct agttagccag
     3121 agagctccca ggctcagatc tggtctaacc agagagaccc gtttatgtat cgagctaggc
     3181 acttaaatac aatatctctg caatgcggca attcagtggt tcgtccaatc catgtcagac
     3241 ccgtctgttg ccttcctaat aaggcacgat cgtaccacct tacttccacc aatcggcatg
     3301 cacggtgctt tttctctcct tgtaaggcat gttgctaact catcgttacc atgttgcaag
     3361 actacaagag tattgcataa gactacatta agcttgcagc tccagctttt gttcccttta
     3421 gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     3481 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     3541 tgcctaatga gtgagctaac tcaccttaat tgcgttgcgc tcactgcccg ctttccagtc
     3601 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3661 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3721 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3781 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3841 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3901 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3961 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4021 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4081 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4141 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4201 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4261 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4321 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4381 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4441 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4501 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4561 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4621 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4681 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4741 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4801 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4861 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4921 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4981 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5041 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5101 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5161 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5221 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5281 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5341 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5401 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5461 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5521 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5581 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
     5641 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     5701 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     5761 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     5821 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     5881 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     5941 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     6001 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     6061 tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     6121 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
     6181 gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca
     6241 ctatagggcg aattgggtac gtccctaggc ctccaaaaaa gcctcctcac tacttctgga
     6301 atagctcaga ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat
     6361 ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg
     6421 ggatagctag agccagacat gataagatac attgatgagt ttggacaaac cacaactaga
     6481 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     6541 attataagct gcaataaaca agttcctctc actctctgat attcatttct ttgcaagtta
     6601 taaatactga ataataagat gacatgaact actactgcta gagattttcc acactgacta
     6661 aaagggtctg agggatctct agttaccaga gtcacacaac agacgggcac acactacttg
     6721 aagcactcaa ggcaagcttt attgaggctt aagcagtggg ttccctagtt agccagagag
     6781 ctcccaggct cagatctggt ctaaccagag agacccagta caagcaaaaa gcagatcttg
     6841 tcttcgttgg gagtgaatta gcccttccag tccccccttt tcttttaaaa agtggctaag
     6901 atctacagct gccttgtaag tcattggtct taaaggtacc gagctcgaat tccaggcggg
     6961 gaggcggccc aaagggagat ccgactcgtc tgagggcgaa ggcgaagacg cggaagaggc
     7021 cgcagagccg gcagcaggcc gcgggaagga aggtccgctg gattgagggc cgaagggacg
     7081 tagcagaagg acgtcccgcg cagaatccag gtggcaacac aggcgagcag ccatggaaag
     7141 gacgtcagct tccccgacaa caccacggaa ttgtcagtgc ccaacagccg agcccctgtc
     7201 cagcagcggg caaggcaggc ggcgatgagt tccgccgtgg caatagggag ggggaaagcg
     7261 aaagtcccgg aaaggagctg acaggtggtg gcaatgcccc aaccagtggg ggttgcgtca
     7321 gcaaacacag tgcacaccac gccacgttgc ctgacaacgg gccacaactc ctcataaaga
     7381 gacagcaacc aggatttata caaggaggag aaaatgaaag ccatacggga agcaatagca
     7441 tgatacaaag gcattaaagc agcgtatcca catagcgtaa aaggagcaac atagttaaga
     7501 ataccagtca atctttcaca aattttgtaa tccagaggtt gattgtcgac tgcagttcga
     7561 acccgggcat atgtctagaa ccggtggatc ggcgctcagc tggaattggc cgctttactt
     7621 gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat
     7681 gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac tgggtgctca ggtagtggtt
     7741 gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag
     7801 ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag ttca
//
