LOCUS       sequence-367458-        7940 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral gene overexpression (backbone/control).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7940)
  AUTHORS   Klusmann
  TITLE     Klusmann Lab Plasmids 2022
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 7940)
  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            "unpublished"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: pLKO5d.SFFV.IRES.dTomato
FEATURES             Location/Qualifiers
     source          1..7940
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             139..372
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    462..865
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(1100..1801)
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(1183..1206)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1381..1400)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1650..1668
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_feature    join(1802..1810,1811..1813,1814..2388)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1975..1994)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     2204..2221
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     promoter        complement(2472..2878)
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     misc_feature    2912..3029
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(3296..3340)
                     /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    3525..3758
                     /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    4251..4376
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             4423..4603
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(4604..4830)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(4858..4878)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4858..4876)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4897..4913)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4897..4913)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4910..4932)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4921..4937
                     /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(4945..4951,4952..4969,4970..4975))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4990..5011
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5128..5145)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5299..5887)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5379..5398)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6058..6849,6850..6918))
                     /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     6681..6700
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6919..7023)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7049..7504)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7186..7207)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7398..7417
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7631..7653
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7645..7662
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7646..7662
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7672..7691
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7672..7690
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      7711..7846
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    join(7886..7940,1..67)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7897..7916)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
       61 acaagttcct ctcactctct gatattcatt tctttgcaag ttataaatac tgaataataa
      121 gatgacatga actactactg ctagagattt tccacactga ctaaaagggt ctgagggatc
      181 tctagttacc agagtcacac aacagacggg cacacactac ttgaagcact caaggcaagc
      241 tttattgagg cttaagcagt gggttcccta gttagccaga gagctcccag gctcagatct
      301 ggtctaacca gagagaccca gtacaagcaa aaagcagatc ttgtcttcgt tgggagttac
      361 gtagcccttc cagtcccccc ttttctttta aaaagtggct aagatctaca gctgccttgt
      421 aagtcattgg tcttaaaggt acctgatatc gaattcgaaa ggacgtcagc ttccccgaca
      481 acaccacgga attatcagtg cccagcaacc tagcccctgt ccagcagcgg gcaaggcagg
      541 cggcgatgag ttctgccgtg gcgatcggga gggggaaagc gaaagtccca gaaaggagtt
      601 gacaggtggt ggcaatgccc cagccagtgg gggttgcgtc agcaaacaca gagcacacca
      661 cgccacgttg acggacaacg ggccacaact cctcataaag agacagcaac caggatttat
      721 acaaggagga gaaaacgaaa gccgtacggg aagcaatagc atgatacaga ggcattaaag
      781 cagcatatcc acacagcgta aaaggagcaa catagttaag aatatcagtc aatctttcac
      841 aaattttgta atccagaggt tgattaacag gaacagagcg taaataacgg gaaagtttct
      901 taacatgttt gtcttgtggc aatacacctg aactagtaat tacatatccc taaaaatgta
      961 aatgattgcc ccaccatttt gttttattaa catttaaatg tatacccaaa tcaagaaaaa
     1021 cagaacaaat atgggaataa atggcggtaa gatgctcgaa ttcctgcagc ccgggggatc
     1081 cgtcgacgcg gccgctttac ttgtacagct cgtccatgcc gtacaggaac aggtggtggc
     1141 ggccctcgga gcgctcgtac tgttccacga tggtgtagtc ctcgttgtgg gaggtgatgt
     1201 ccagcttggt gtccacgtag tagtagccgg gcagttgcac gggcttcttg gccatgtaga
     1261 tggtcttgaa ctccaccagg tagtggccgc cgtccttcag cttcagggcc tggtggatct
     1321 cgcccttcag cacgccgtcg cgggggtaca ggcgctcggt ggaggcctcc cagcccatgg
     1381 tcttcttctg cattacgggg ccgtcggggg ggaagttggt gccgcgcatc ttcaccttgt
     1441 agatcagcgt gccgtcctgc agggaggagt cctgggtcac ggtcaccaga ccgccgtcct
     1501 cgaagttcat cacgcgctcc cacttgaagc cctcggggaa ggacagcttc ttgtaatcgg
     1561 ggatgtcggc ggggtgcttc acgtacgcct tggagccgta catgaactgg ggggacagga
     1621 tgtcccaggc gaagggcagg gggccgccct tggtcacctt cagcttggcg gtctgggtgc
     1681 cctcgtaggg gcggccctcg ccctcgccct cgatctcgaa ctcgtggccg ttcatggagc
     1741 cctccatgcg caccttgaag cgcatgaact ctttgatgac ctcctcgccc ttgctcacca
     1801 tggttgtggc catattatca tcgtgttttt caaaggaaaa ccacgtcccc gtggttcggg
     1861 gggcctagac gtttttttaa cctcgactaa acacatgtaa agcatgtgca ccgaggcccc
     1921 agatcagatc ccatacaatg gggtaccttc tgggcatcct tcagcccctt gttgaatacg
     1981 cttgaggaga gccatttgac tctttccaca actatccaac tcacaacgtg gcactggggt
     2041 tgtgccgcct ttgcaggtgt atcttataca cgtggctttt ggccgcagag gcacctgtcg
     2101 ccaggtgggg ggttccgctg cctgcaaagg gtcgctacag acgttgtttg tcttcaagaa
     2161 gcttccagag gaactgcttc cttcacgaca ttcaacagac cttgcattcc tttggcgaga
     2221 ggggaaagac ccctaggaat gctcgtcaag aagacagggc caggtttccg ggccctcaca
     2281 ttgccaaaag acggcaatat ggtggaaaat aacatataga caaacgcaca ccggccttat
     2341 tccaagcggc ttcggccagt aacgttaggg gggggggagg gagaggggcg gatcctcagt
     2401 tactcatgca tacgcgccgg agtatactag taccggtgga tccgtcgacg gtatcgataa
     2461 gcttgattcg accggccgac tcagtctgtc ggaggactgg cgcgccgagt gaggggttgt
     2521 gagctctttt atagagctcg ggaagcagaa gcgcgcgaac agaagcgaga agcaggctga
     2581 ttggttaatt caaataaggc gcagggtcat ttcaggtcct tgggggagcc tggaaacatc
     2641 tgatgggtct taagaaactg ctgagggttg ggccatatct ggggaccatc tgttcttggc
     2701 ctcgggccgg ggccgaaact gcggtgacca tctgttcttg gccccgggcc ggggccgaaa
     2761 ctgctcaccg cagatatcct gtttggccca acgttagcta ttttcatgta cccgcccttg
     2821 atctgaactt ctctattctt ggtttggtat ttttccatgc cttgcaaaat ggcgttactg
     2881 cagctagctc gaggctagtc tcgtgatcga taaaattttg aatttttgta atttgttttt
     2941 gtaattcttt agtttgtatg tctgttgcta ttatgtctac tattctttcc cctgcactgt
     3001 accccccaat cccccctttt cttttaaaag ttaaccgata ccgtcgagat ccgttcacta
     3061 atcgaatgga tctgtctctg tctctctctc caccttcttc ttctattcct tcgggcctgt
     3121 cgggtcccct cggggttggg aggtgggtct gaaacgataa tggtgaatat ccctgcctaa
     3181 ctctattcac tatagaaagt acagcaaaaa ctattcttaa acctaccaag cctcctacta
     3241 tcattatgaa taattttata taccacagcc aatttgttat gttaaaccaa ttccacaaac
     3301 ttgcccattt atctaattcc aataattctt gttcattctt ttcttgctgg ttttgcgatt
     3361 cttcaattaa ggagtgtatt aagcttgtgt aattgttaat ttctctgtcc cactccatcc
     3421 aggtcgtgtg attccaaatc tgttccagag atttattact ccaactagca ttccaaggca
     3481 cagcagtggt gcaaatgagt tttccagagc aaccccaaat ccccaggagc tgttgatcct
     3541 ttaggtatct ttccacagcc aggattcttg cctggagctg cttgatgccc cagactgtga
     3601 gttgcaacag atgctgttgc gcctcaatag ccctcagcaa attgttctgc tgctgcacta
     3661 taccagacaa taattgtctg gcctgtaccg tcagcgtcat tgacgctgcg cccatagtgc
     3721 ttcctgctgc tcccaagaac ccaaggaaca aagctcctat tcccactgct cttttttctc
     3781 tctgcaccac tcttctcttt gccttggtgg gtgctactcc taatggttca atttttacta
     3841 ctttatattt atataattca cttctccaat tgtccctcat atctcctcct ccaggtctga
     3901 agatcagcgg ccgcttgctg tgcggtggtc ttacttttgt tttgctcttc ctctatcttg
     3961 tctaaagctt ccttggtgtc ttttatctct atcctttgat gcacacaata gagggttgct
     4021 actgtattat ataatgatct aagttcttct gatcctgtct gaagggatgg ttgtagctgt
     4081 cccagtattt gtctacagcc ttctgatgtt tctaacaggc caggattaac tgcgaatcgt
     4141 tctagctccc tgcttgccca tactatatgt tttaatttat attttttctt tccccctggc
     4201 cttaaccgaa ttttttccca tcgcgatcta attctccccc gcttaatact gacgctctcg
     4261 cacccatctc tctccttcta gcctccgcta gtcaaaattt ttggcgtact caccagtcgc
     4321 cgcccctcgc ctcttgccgt gcgcgcttca gcaagccgag tcctgcgtcg agagagctcc
     4381 tctggtttcc ctttcgcttt caagtccctg ttcgggcgcc actgctagag attttccaca
     4441 ctgactaaaa gggtctgagg gatctctagt taccagagtc acacaacaga cgggcacaca
     4501 ctacttgaag cactcaaggc aagctttatt gaggcttaag cagtgggttc cctagttagc
     4561 cagagagctc ccaggctcag atctggtcta accagagaga cccgtttatg tatcgagcta
     4621 ggcacttaaa tacaatatct ctgcaatgcg gcaattcagt ggttcgtcca atccatgtca
     4681 gacccgtctg ttgccttcct aataaggcac gatcgtacca ccttacttcc accaatcggc
     4741 atgcacggtg ctttttctct ccttgtaagg catgttgcta actcatcgtt accatgttgc
     4801 aagactacaa gagtattgca taagactaca ttaagcttgc agctccagct tttgttccct
     4861 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4921 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4981 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5041 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5101 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5161 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5221 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5281 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5341 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5401 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5461 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5521 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5581 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5641 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5701 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5761 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5821 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5881 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5941 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6001 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6061 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6121 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6181 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6241 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6301 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6361 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6421 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6481 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6541 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6601 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6661 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6721 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6781 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6841 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6901 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6961 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7021 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     7081 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     7141 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     7201 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     7261 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     7321 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     7381 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     7441 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     7501 gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     7561 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     7621 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac
     7681 tcactatagg gcgaattggg tacgtcccta ggcctccaaa aaagcctcct cactacttct
     7741 ggaatagctc agaggccgag gcggcctcgg cctctgcata aataaaaaaa attagtcagc
     7801 catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg
     7861 gcgggatagc tagagccaga catgataaga tacattgatg agtttggaca aaccacaact
     7921 agaatgcagt gaaaaaaatg
//
