LOCUS       sequence-364224-        7322 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct for expression of tagRFP-T targeted to 
            mitochondria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7322)
  AUTHORS   Greiner D, Scott TM, Olson GS, Aderem A, Roh-Johnson M, Johnson JS
  TITLE     Genetic Modification of Primary Human Myeloid Cells to Study Cell 
            Migration, Activation, and Organelle Dynamics.
  JOURNAL   Curr Protoc. 2022 Aug;2(8):e514. doi: 10.1002/cpz1.514.
  PUBMED    36018279
REFERENCE   2  (bases 1 to 7322)
  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: "Curr 
            Protoc."; date: "2022-08"; volume: "2(8):e514. doi"; pages: " 
            10.1002/cpz1.514"
COMMENT     Sequence Label: pLKO-mito-tagRFP
FEATURES             Location/Qualifiers
     source          1..7322
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(259..363)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(389..844)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(526..547)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     738..757
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     971..993
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     985..1002
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     986..1002
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1012..1031
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1012..1030
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      1051..1186
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1226..1347
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1237..1256)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1291..1310
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             1419..1652
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     CDS             complement(1806..2537)
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTETLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     regulatory      2534..2543
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2571..2645)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     promoter        complement(2673..3183)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2736..2754)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    3232..3349
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(3412..3660)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3470..3489)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3640..3660)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(3958..4002)
                     /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    4187..4420
                     /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    4913..5038
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             5085..5265
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(5266..5492)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(5520..5540)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5520..5538)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5559..5575)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5559..5575)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5572..5594)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5583..5599
                     /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(5607..5613,5614..5631,5632..5637))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5652..5673
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5790..5807)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5961..6549)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6041..6060)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6720..7322,1..189,190..258))
                     /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"
ORIGIN
        1 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
       61 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
      121 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
      181 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
      241 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
      301 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      361 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
      421 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
      481 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
      541 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
      601 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
      661 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
      721 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
      781 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
      841 gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
      901 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
      961 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac
     1021 tcactatagg gcgaattggg tacgtcccta ggcctccaaa aaagcctcct cactacttct
     1081 ggaatagctc agaggccgag gcggcctcgg cctctgcata aataaaaaaa attagtcagc
     1141 catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg
     1201 gcgggatagc tagagccaga catgataaga tacattgatg agtttggaca aaccacaact
     1261 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     1321 accattataa gctgcaataa acaagttcct ctcactctct gatattcatt tctttgcaag
     1381 ttataaatac tgaataataa gatgacatga actactactg ctagagattt tccacactga
     1441 ctaaaagggt ctgagggatc tctagttacc agagtcacac aacagacggg cacacactac
     1501 ttgaagcact caaggcaagc tttattgagg cttaagcagt gggttcccta gttagccaga
     1561 gagctcccag gctcagatct ggtctaacca gagagaccca gtacaagcaa aaagcagatc
     1621 ttgtcttcgt tgggagtgaa ttagcccttc cagtcccccc ttttctttta aaaagtggct
     1681 aagatctaca gctgccttgt aagtcattgg tcttaaaggt accgatgcat ggggtcgtgc
     1741 gctcctttcg gtcgggcgct gcgggtcgtg gggcgggcgt tacttgtaca gatatccgta
     1801 cgttacttgt acagctcgtc catgccatta agtttgtgcc ccagtttgct agggaggtcg
     1861 cagtatctgg ccacagccac ctcgtgctgc tcgacgtagg tctctttgtc ggcctccttg
     1921 attctttcca gtctgtggtc cacatagtag acgccgggca tcttgaggtt cttagcgggt
     1981 ttcttggatc tgtatgtggt cttgaagttg cagatcaggt ggcccccgcc cacgagcttc
     2041 agggccatgt cggttctgcc ttccaggccg ccgtcagcgg ggtacagggt ctcggtgttg
     2101 gcctcccagc cgagtgtttt cttctgcatc acagggccgt tggatgggaa gttcacccct
     2161 ctgatcttga cgttgtagat gaggcagccg tcctggaggc tggtgtcctg ggtagcggtc
     2221 agcacgcccc cgtcttcgta tgtggtgact ctctcccatg tgaagccctc agggaaggac
     2281 tgcttaaaga aatcggggat gccctgggtg tggttgatga aggttctgct gccgtacatg
     2341 aagctggtag ccaggatgtc gaaggcgaag gggagagggc cgccctcgac caccttgatt
     2401 ctcatggtct gggtgccctc gtagggcttg ccttcgccct cggatgtgca cttgaagtgg
     2461 tggttgttca cggtgccctc catgtacagc ttcatgtgca tgttctcctt aatcagctct
     2521 tcgcccttag acaccatggt ggcgaccggt ggatccccca acgaatggat cttggcgcgc
     2581 ggcactggga gccgccgggc cgagcctgtc aagccccgca gcagcagcgg cgtcaggacg
     2641 gacatggtaa gctccggtga cgtcggatcc ccctggggag agaggtcggt gattcggtca
     2701 acgagggagc cgactgccga cgtgcgctcc ggaggcttgc agaatgcgga acaccgcgcg
     2761 ggcaggaaca gggcccacac taccgcccca caccccgcct cccgcaccgc cccttcccgg
     2821 ccgctgctct cggcgcgccc tgctgagcag ccgctattgg ccacagccca tcgcggtcgg
     2881 cgcgctgcca ttgctccctg gcgctgtccg tctgcgaggg tactagtgag acgtgcggct
     2941 tccgtttgtc acgtccggca cgccgcgaac cgcaaggaac cttcccgact taggggcgga
     3001 gcaggaagcg tcgccggggg gcccacaagg gtagcggcga agatccgggt gacgctgcga
     3061 acggacgtga agaatgtgcg agacccaggg tcggcgccgc tgcgtttccc ggaaccacgc
     3121 ccagagcagc cgcgtccctg cgcaaaccca gggctgcctt ggaaaaggcg caaccccaac
     3181 cccctcgagc cgcggccaaa gtggatctct gctgtccctg taataaaccc gaaaattttg
     3241 aatttttgta atttgttttt gtaattcttt agtttgtatg tctgttgcta ttatgtctac
     3301 tattctttcc cctgcactgt accccccaat cccccctttt cttttaaaat tgtggatgaa
     3361 tactgccatt tgtctcgagg tcgagaattc acgcgtgcat acctgcggac cggtgtttcg
     3421 tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat
     3481 atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt
     3541 tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttccaattat
     3601 ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc
     3661 ggtgaagggg gcggccgctc gaggctagtc tcgtgatcga taccgtcgag atccgttcac
     3721 taatcgaatg gatctgtctc tgtctctctc tccaccttct tcttctattc cttcgggcct
     3781 gtcgggtccc ctcggggttg ggaggtgggt ctgaaacgat aatggtgaat atccctgcct
     3841 aactctattc actatagaaa gtacagcaaa aactattctt aaacctacca agcctcctac
     3901 tatcattatg aataatttta tataccacag ccaatttgtt atgttaaacc aattccacaa
     3961 acttgcccat ttatctaatt ccaataattc ttgttcattc ttttcttgct ggttttgcga
     4021 ttcttcaatt aaggagtgta ttaagcttgt gtaattgtta atttctctgt cccactccat
     4081 ccaggtcgtg tgattccaaa tctgttccag agatttatta ctccaactag cattccaagg
     4141 cacagcagtg gtgcaaatga gttttccaga gcaaccccaa atccccagga gctgttgatc
     4201 ctttaggtat ctttccacag ccaggattct tgcctggagc tgcttgatgc cccagactgt
     4261 gagttgcaac agatgctgtt gcgcctcaat agccctcagc aaattgttct gctgctgcac
     4321 tataccagac aataattgtc tggcctgtac cgtcagcgtc attgacgctg cgcccatagt
     4381 gcttcctgct gctcccaaga acccaaggaa caaagctcct attcccactg ctcttttttc
     4441 tctctgcacc actcttctct ttgccttggt gggtgctact cctaatggtt caatttttac
     4501 tactttatat ttatataatt cacttctcca attgtccctc atatctcctc ctccaggtct
     4561 gaagatcagc ggccgcttgc tgtgcggtgg tcttactttt gttttgctct tcctctatct
     4621 tgtctaaagc ttccttggtg tcttttatct ctatcctttg atgcacacaa tagagggttg
     4681 ctactgtatt atataatgat ctaagttctt ctgatcctgt ctgaagggat ggttgtagct
     4741 gtcccagtat ttgtctacag ccttctgatg tttctaacag gccaggatta actgcgaatc
     4801 gttctagctc cctgcttgcc catactatat gttttaattt atattttttc tttccccctg
     4861 gccttaaccg aattttttcc catcgcgatc taattctccc ccgcttaata ctgacgctct
     4921 cgcacccatc tctctccttc tagcctccgc tagtcaaaat ttttggcgta ctcaccagtc
     4981 gccgcccctc gcctcttgcc gtgcgcgctt cagcaagccg agtcctgcgt cgagagagct
     5041 cctctggttt ccctttcgct ttcaagtccc tgttcgggcg ccactgctag agattttcca
     5101 cactgactaa aagggtctga gggatctcta gttaccagag tcacacaaca gacgggcaca
     5161 cactacttga agcactcaag gcaagcttta ttgaggctta agcagtgggt tccctagtta
     5221 gccagagagc tcccaggctc agatctggtc taaccagaga gacccgttta tgtatcgagc
     5281 taggcactta aatacaatat ctctgcaatg cggcaattca gtggttcgtc caatccatgt
     5341 cagacccgtc tgttgccttc ctaataaggc acgatcgtac caccttactt ccaccaatcg
     5401 gcatgcacgg tgctttttct ctccttgtaa ggcatgttgc taactcatcg ttaccatgtt
     5461 gcaagactac aagagtattg cataagacta cattaagctt gcagctccag cttttgttcc
     5521 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     5581 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     5641 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     5701 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5761 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5821 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5881 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5941 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6001 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6061 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6121 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6181 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6241 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6301 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6361 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6421 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6481 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6541 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6601 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6661 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6721 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6781 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6841 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6901 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6961 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7021 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7081 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7141 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7201 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7261 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7321 tc
//
