LOCUS       sequence-364209-        7307 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct for expression of mCerulean 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-mCer
FEATURES             Location/Qualifiers
     source          1..7307
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(104..123)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      917..1505
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1406..1425
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1659..1676
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1793..1814
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1829..1834,1835..1852,1853..1859)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1867..1883
                     /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     1872..1894
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1891..1907
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1891..1907
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1926..1946
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1928..1946
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1974..2200
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             2201..2381
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2428..2553
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3046..3279
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3464..3508
                     /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        3806..4054
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3806..3826
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3977..3996
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    4117..4234
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4283..4793
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4712..4730
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4821..4895
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      4923..4932
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4929..4931,4932..4934,4935..5648)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4974..4995)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5235..5254)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5582..5603
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             5799..6032
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6104..6225
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6141..6160)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6195..6214
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6265..6400
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6420..6439)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6421..6439)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6449..6466)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6449..6465)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6458..6480)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6607..7062
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6694..6713)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6904..6925
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7088..7192
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7193..7261,7262..7307,1..746)
                     /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 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
       61 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      121 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      181 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      241 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      301 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      361 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      421 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      481 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      541 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      601 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      661 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      721 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
      781 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
      841 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
      901 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      961 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1021 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     1081 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1141 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1201 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1261 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1321 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1381 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1441 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1501 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1561 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1621 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     1681 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     1741 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     1801 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     1861 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     1921 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc
     1981 ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca
     2041 aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt
     2101 attaggaagg caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg
     2161 cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct ggttagacca
     2221 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     2281 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     2341 atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac
     2401 ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg
     2461 cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga
     2521 ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg
     2581 cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata
     2641 gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca
     2701 gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa
     2761 cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata
     2821 aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc
     2881 gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag
     2941 aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa
     3001 ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct
     3061 tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca
     3121 ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga
     3181 ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat
     3241 cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg
     3301 aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga
     3361 acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag
     3421 cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt
     3481 attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg
     3541 gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc
     3601 tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca
     3661 cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag
     3721 agacagagac agatccattc gattagtgaa cggatctcga cggtatcgat cacgagacta
     3781 gcctcgagcg gccgccccct tcaccgaggg cctatttccc atgattcctt catatttgca
     3841 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     3901 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     3961 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     4021 ggctttatat atcttgtgga aaggacgaaa caccggtccg caggtatgca cgcgtgaatt
     4081 ctcgacctcg agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg
     4141 ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga
     4201 attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga
     4261 tccactttgg ccgcggctcg agggggttgg ggttgcgcct tttccaaggc agccctgggt
     4321 ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa acgcagcggc gccgaccctg
     4381 ggtctcgcac attcttcacg tccgttcgca gcgtcacccg gatcttcgcc gctacccttg
     4441 tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg
     4501 cggcgtgccg gacgtgacaa acggaagccg cacgtctcac tagtaccctc gcagacggac
     4561 agcgccaggg agcaatggca gcgcgccgac cgcgatgggc tgtggccaat agcggctgct
     4621 cagcagggcg cgccgagagc agcggccggg aaggggcggt gcgggaggcg gggtgtgggg
     4681 cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag cctccggagc
     4741 gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga cctctctccc cagggggatc
     4801 cgacgtcacc ggagcttacc atgtccgtcc tgacgccgct gctgctgcgg ggcttgacag
     4861 gctcggcccg gcggctccca gtgccgcgcg ccaagatcca ttcgttgttc gaaccaccgg
     4921 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     4981 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     5041 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     5101 ggcccaccct cgtgaccacc ctgagctggg gcgtgcagtg cttcgcccgc taccccgacc
     5161 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     5221 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     5281 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     5341 tggggcacaa gctggagtac aacgccatcc acggcaacgt ctatatcacc gccgacaagc
     5401 agaagaacgg catcaaggcc aacttcggcc tcaactgcaa catcgaggac ggcagcgtgc
     5461 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     5521 acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc
     5581 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     5641 acaagtaacg tacggatatc tgtacaagta acgcccgccc cacgacccgc agcgcccgac
     5701 cgaaaggagc gcacgacccc atgcatcggt acctttaaga ccaatgactt acaaggcagc
     5761 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     5821 acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc
     5881 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
     5941 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
     6001 acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc ttattattca
     6061 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc
     6121 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     6181 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta
     6241 gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     6301 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     6361 gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc
     6421 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     6481 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     6541 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     6601 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     6661 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     6721 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     6781 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     6841 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     6901 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     6961 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     7021 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     7081 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     7141 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     7201 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     7261 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgg
//
