LOCUS       sequence-364227-        7223 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct for expression of tagRFP-T targeted to 
            peroxisomes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7223)
  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 7223)
  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-perox-tagRFP
FEATURES             Location/Qualifiers
     source          1..7223
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(105..124)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      918..1506
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1407..1426
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1660..1677
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1794..1815
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1830..1835,1836..1853,1854..1860)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1868..1884
                     /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     1873..1895
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1892..1908
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1892..1908
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1927..1947
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1929..1947
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1975..2201
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             2202..2382
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2429..2554
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3047..3280
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3465..3509
                     /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        3807..4055
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3807..3827
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3978..3997
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    4118..4235
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4284..4794
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4713..4731
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4822..5553
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTETLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     LTR             5716..5949
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6021..6142
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6058..6077)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6112..6131
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6182..6317
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6337..6356)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6338..6356)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6366..6383)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6366..6382)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6375..6397)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6524..6979
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6611..6630)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6821..6842
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7005..7109
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7110..7178,7179..7223,1..747)
                     /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 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
       61 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
      121 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
      181 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
      241 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
      301 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
      361 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
      421 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
      481 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
      541 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
      601 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
      661 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
      721 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
      781 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
      841 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
      901 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
      961 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1021 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     1081 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1141 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     1201 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     1261 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     1321 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     1381 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     1441 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     1501 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     1561 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     1621 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     1681 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     1741 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     1801 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     1861 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     1921 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt
     1981 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac
     2041 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct
     2101 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt
     2161 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc
     2221 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     2281 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     2341 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     2401 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     2461 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     2521 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     2581 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     2641 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     2701 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     2761 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     2821 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     2881 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     2941 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     3001 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     3061 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     3121 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     3181 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     3241 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     3301 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     3361 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     3421 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     3481 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     3541 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     3601 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     3661 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     3721 gagacagaga cagatccatt cgattagtga acggatctcg acggtatcga tcacgagact
     3781 agcctcgagc ggccgccccc ttcaccgagg gcctatttcc catgattcct tcatatttgc
     3841 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     3901 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     3961 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     4021 tggctttata tatcttgtgg aaaggacgaa acaccggtcc gcaggtatgc acgcgtgaat
     4081 tctcgacctc gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg
     4141 gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag
     4201 aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag
     4261 atccactttg gccgcggctc gagggggttg gggttgcgcc ttttccaagg cagccctggg
     4321 tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg cgccgaccct
     4381 gggtctcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt
     4441 gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc cttgcggttc
     4501 gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct cgcagacgga
     4561 cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc
     4621 tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg
     4681 gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa gcctccggag
     4741 cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc ccagggggat
     4801 ccgacgtcac cggagcttac catggtgtct aagggcgaag agctgattaa ggagaacatg
     4861 cacatgaagc tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag
     4921 ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc
     4981 cctctcccct tcgccttcga catcctggct accagcttca tgtacggcag cagaaccttc
     5041 atcaaccaca cccagggcat ccccgatttc tttaagcagt ccttccctga gggcttcaca
     5101 tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc
     5161 ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt cccatccaac
     5221 ggccctgtga tgcagaagaa aacactcggc tgggaggcca acaccgagac cctgtacccc
     5281 gctgacggcg gcctggaagg cagaaccgac atggccctga agctcgtggg cgggggccac
     5341 ctgatctgca acttcaagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg
     5401 cccggcgtct actatgtgga ccacagactg gaaagaatca aggaggccga caaagagacc
     5461 tacgtcgagc agcacgaggt ggctgtggcc agatactgcg acctccctag caaactgggg
     5521 cacaaactta atggcatgga cgagctgtac aagtccaagc tgtaacgtac ggatatctgt
     5581 acaagtaacg cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg
     5641 catcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     5701 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     5761 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     5821 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     5881 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     5941 tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     6001 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca
     6061 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     6121 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc
     6181 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     6241 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     6301 aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg
     6361 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     6421 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     6481 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     6541 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     6601 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     6661 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     6721 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     6781 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     6841 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     6901 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     6961 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     7021 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     7081 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     7141 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     7201 taaaagatgc tgaagatcag ttg
//
