LOCUS       sequence-364239-        7307 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct for expression of mEmerald targeted to 
            mitochondria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7307)
  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 7307)
  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-mEm
FEATURES             Location/Qualifiers
     source          1..7307
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     433..452
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(671..775)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(801..1256)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(938..959)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1150..1169
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1383..1405
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1397..1414
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1398..1414
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1424..1443
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1424..1442
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      1463..1598
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1638..1759
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1649..1668)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1703..1722
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             1831..2064
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     CDS             complement(join(2215..2928,2929..2931,2932..2934))
                     /codon_start=1
                     /product="brightly fluorescent monomeric variant of GFP"
                     /label=mEmerald
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2260..2281)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2609..2628
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2868..2889
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2931..2940
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2968..3042)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     promoter        complement(3070..3580)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3133..3151)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    3629..3746
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(3809..4057)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3867..3886)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4037..4057)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(4355..4399)
                     /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    4584..4817
                     /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    5310..5435
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             5482..5662
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(5663..5889)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(5917..5937)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5917..5935)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5956..5972)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5956..5972)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5969..5991)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5980..5996
                     /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(6004..6010,6011..6028,6029..6034))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6049..6070
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6187..6204)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6358..6946)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6438..6457)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7117..7307,1..601,602..670))
                     /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 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
       61 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
      121 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
      181 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
      241 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
      301 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
      361 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
      421 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
      481 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
      541 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
      601 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
      661 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
      721 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
      781 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
      841 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
      901 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
      961 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     1021 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     1081 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     1141 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     1201 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     1261 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     1321 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     1381 ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg actcactata
     1441 gggcgaattg ggtacgtccc taggcctcca aaaaagcctc ctcactactt ctggaatagc
     1501 tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc
     1561 ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggata
     1621 gctagagcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     1681 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     1741 aagctgcaat aaacaagttc ctctcactct ctgatattca tttctttgca agttataaat
     1801 actgaataat aagatgacat gaactactac tgctagagat tttccacact gactaaaagg
     1861 gtctgaggga tctctagtta ccagagtcac acaacagacg ggcacacact acttgaagca
     1921 ctcaaggcaa gctttattga ggcttaagca gtgggttccc tagttagcca gagagctccc
     1981 aggctcagat ctggtctaac cagagagacc cagtacaagc aaaaagcaga tcttgtcttc
     2041 gttgggagtg aattagccct tccagtcccc ccttttcttt taaaaagtgg ctaagatcta
     2101 cagctgcctt gtaagtcatt ggtcttaaag gtaccgatgc atggggtcgt gcgctccttt
     2161 cggtcgggcg ctgcgggtcg tggggcgggc gttacttgta cagatatccg tacgttactt
     2221 gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat
     2281 gtgatcgcgc ttctcgttgg ggtctttgct cagcttggac tgggtgctca ggtagtggtt
     2341 gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag
     2401 ctgcacgctg ccgtcctcga tgttgtggcg ggtcttgaag ttcaccttga tgccgttctt
     2461 ctgcttgtcg gcggtgatat agaccttgtg gctgttgtag ttgtactcca gcttgtgccc
     2521 caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt
     2581 gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat
     2641 ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat
     2701 gtggtcgggg tagcgggcga agcactgcac gccgtaggtc aaggtggtca cgagggtggg
     2761 ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt
     2821 ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag
     2881 ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca ccatggtggc
     2941 gaccggtgga tcccccaacg aatggatctt ggcgcgcggc actgggagcc gccgggccga
     3001 gcctgtcaag ccccgcagca gcagcggcgt caggacggac atggtaagct ccggtgacgt
     3061 cggatccccc tggggagaga ggtcggtgat tcggtcaacg agggagccga ctgccgacgt
     3121 gcgctccgga ggcttgcaga atgcggaaca ccgcgcgggc aggaacaggg cccacactac
     3181 cgccccacac cccgcctccc gcaccgcccc ttcccggccg ctgctctcgg cgcgccctgc
     3241 tgagcagccg ctattggcca cagcccatcg cggtcggcgc gctgccattg ctccctggcg
     3301 ctgtccgtct gcgagggtac tagtgagacg tgcggcttcc gtttgtcacg tccggcacgc
     3361 cgcgaaccgc aaggaacctt cccgacttag gggcggagca ggaagcgtcg ccggggggcc
     3421 cacaagggta gcggcgaaga tccgggtgac gctgcgaacg gacgtgaaga atgtgcgaga
     3481 cccagggtcg gcgccgctgc gtttcccgga accacgccca gagcagccgc gtccctgcgc
     3541 aaacccaggg ctgccttgga aaaggcgcaa ccccaacccc ctcgagccgc ggccaaagtg
     3601 gatctctgct gtccctgtaa taaacccgaa aattttgaat ttttgtaatt tgtttttgta
     3661 attctttagt ttgtatgtct gttgctatta tgtctactat tctttcccct gcactgtacc
     3721 ccccaatccc cccttttctt ttaaaattgt ggatgaatac tgccatttgt ctcgaggtcg
     3781 agaattcacg cgtgcatacc tgcggaccgg tgtttcgtcc tttccacaag atatataaag
     3841 ccaagaaatc gaaatacttt caagttacgg taagcatatg atagtccatt ttaaaacata
     3901 attttaaaac tgcaaactac ccaagaaatt attactttct acgtcacgta ttttgtacta
     3961 atatctttgt gtttacagtc aaattaattc caattatctc tctaacagcc ttgtatcgta
     4021 tatgcaaata tgaaggaatc atgggaaata ggccctcggt gaagggggcg gccgctcgag
     4081 gctagtctcg tgatcgatac cgtcgagatc cgttcactaa tcgaatggat ctgtctctgt
     4141 ctctctctcc accttcttct tctattcctt cgggcctgtc gggtcccctc ggggttggga
     4201 ggtgggtctg aaacgataat ggtgaatatc cctgcctaac tctattcact atagaaagta
     4261 cagcaaaaac tattcttaaa cctaccaagc ctcctactat cattatgaat aattttatat
     4321 accacagcca atttgttatg ttaaaccaat tccacaaact tgcccattta tctaattcca
     4381 ataattcttg ttcattcttt tcttgctggt tttgcgattc ttcaattaag gagtgtatta
     4441 agcttgtgta attgttaatt tctctgtccc actccatcca ggtcgtgtga ttccaaatct
     4501 gttccagaga tttattactc caactagcat tccaaggcac agcagtggtg caaatgagtt
     4561 ttccagagca accccaaatc cccaggagct gttgatcctt taggtatctt tccacagcca
     4621 ggattcttgc ctggagctgc ttgatgcccc agactgtgag ttgcaacaga tgctgttgcg
     4681 cctcaatagc cctcagcaaa ttgttctgct gctgcactat accagacaat aattgtctgg
     4741 cctgtaccgt cagcgtcatt gacgctgcgc ccatagtgct tcctgctgct cccaagaacc
     4801 caaggaacaa agctcctatt cccactgctc ttttttctct ctgcaccact cttctctttg
     4861 ccttggtggg tgctactcct aatggttcaa tttttactac tttatattta tataattcac
     4921 ttctccaatt gtccctcata tctcctcctc caggtctgaa gatcagcggc cgcttgctgt
     4981 gcggtggtct tacttttgtt ttgctcttcc tctatcttgt ctaaagcttc cttggtgtct
     5041 tttatctcta tcctttgatg cacacaatag agggttgcta ctgtattata taatgatcta
     5101 agttcttctg atcctgtctg aagggatggt tgtagctgtc ccagtatttg tctacagcct
     5161 tctgatgttt ctaacaggcc aggattaact gcgaatcgtt ctagctccct gcttgcccat
     5221 actatatgtt ttaatttata ttttttcttt ccccctggcc ttaaccgaat tttttcccat
     5281 cgcgatctaa ttctcccccg cttaatactg acgctctcgc acccatctct ctccttctag
     5341 cctccgctag tcaaaatttt tggcgtactc accagtcgcc gcccctcgcc tcttgccgtg
     5401 cgcgcttcag caagccgagt cctgcgtcga gagagctcct ctggtttccc tttcgctttc
     5461 aagtccctgt tcgggcgcca ctgctagaga ttttccacac tgactaaaag ggtctgaggg
     5521 atctctagtt accagagtca cacaacagac gggcacacac tacttgaagc actcaaggca
     5581 agctttattg aggcttaagc agtgggttcc ctagttagcc agagagctcc caggctcaga
     5641 tctggtctaa ccagagagac ccgtttatgt atcgagctag gcacttaaat acaatatctc
     5701 tgcaatgcgg caattcagtg gttcgtccaa tccatgtcag acccgtctgt tgccttccta
     5761 ataaggcacg atcgtaccac cttacttcca ccaatcggca tgcacggtgc tttttctctc
     5821 cttgtaaggc atgttgctaa ctcatcgtta ccatgttgca agactacaag agtattgcat
     5881 aagactacat taagcttgca gctccagctt ttgttccctt tagtgagggt taattgcgcg
     5941 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     6001 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     6061 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6121 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     6181 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     6241 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     6301 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     6361 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     6421 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6481 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6541 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6601 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6661 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6721 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6781 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6841 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6901 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6961 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     7021 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     7081 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     7141 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     7201 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     7261 cccacgctca ccggctccag atttatcagc aataaaccag ccagccg
//
