LOCUS       sequence-364230-        7199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct for expression of mEmerald.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7199)
  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 7199)
  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-cell-mEm
FEATURES             Location/Qualifiers
     source          1..7199
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(102..121)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      915..1503
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1404..1423
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1657..1674
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1791..1812
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1827..1832,1833..1850,1851..1857)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1865..1881
                     /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     1870..1892
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1889..1905
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1889..1905
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1924..1944
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1926..1944
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1972..2198
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             2199..2379
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2426..2551
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3044..3277
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3462..3506
                     /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        3804..4052
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3804..3824
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3975..3994
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    4115..4232
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4281..4791
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4710..4728
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             join(4819..4821,4822..4824,4825..5538)
                     /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(4864..4885)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5125..5144)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5472..5493
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             5689..5922
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5994..6115
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6031..6050)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6085..6104
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6155..6290
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6310..6329)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6311..6329)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6339..6356)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6339..6355)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6348..6370)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6497..6952
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6584..6603)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6794..6815
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6978..7082
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7083..7151,7152..7199,1..744)
                     /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 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
       61 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
      121 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
      181 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
      241 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
      301 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
      361 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
      421 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
      481 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
      541 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
      601 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
      661 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
      721 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
      781 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
      841 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
      901 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
      961 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     1021 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     1081 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     1141 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     1201 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     1261 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     1321 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     1381 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     1441 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     1501 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     1561 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     1621 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     1681 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     1741 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     1801 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     1861 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     1921 cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt
     1981 atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag
     2041 gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat
     2101 taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca
     2161 gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga
     2221 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     2281 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     2341 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
     2401 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
     2461 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
     2521 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
     2581 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
     2641 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
     2701 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     2761 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     2821 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     2881 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     2941 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     3001 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     3061 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     3121 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     3181 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     3241 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     3301 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     3361 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     3421 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     3481 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     3541 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     3601 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc
     3661 tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag
     3721 acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgatca cgagactagc
     3781 ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata
     3841 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
     3901 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     3961 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     4021 ctttatatat cttgtggaaa ggacgaaaca ccggtccgca ggtatgcacg cgtgaattct
     4081 cgacctcgag acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg
     4141 ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat
     4201 tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc
     4261 cactttggcc gcggctcgag ggggttgggg ttgcgccttt tccaaggcag ccctgggttt
     4321 gcgcagggac gcggctgctc tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg
     4381 tctcgcacat tcttcacgtc cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg
     4441 ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg
     4501 gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta gtaccctcgc agacggacag
     4561 cgccagggag caatggcagc gcgccgaccg cgatgggctg tggccaatag cggctgctca
     4621 gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg
     4681 gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc
     4741 acgtcggcag tcggctccct cgttgaccga atcaccgacc tctctcccca gggggatccg
     4801 acgtcaccgg agcttaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     4861 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     4921 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     4981 cccgtgccct ggcccaccct cgtgaccacc ttgacctacg gcgtgcagtg cttcgcccgc
     5041 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     5101 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     5161 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     5221 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaaggt ctatatcacc
     5281 gccgacaagc agaagaacgg catcaaggtg aacttcaaga cccgccacaa catcgaggac
     5341 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     5401 ctgctgcccg acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag
     5461 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     5521 gacgagctgt acaagtaacg tacggatatc tgtacaagta acgcccgccc cacgacccgc
     5581 agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga ccaatgactt
     5641 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     5701 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     5761 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     5821 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     5881 gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc
     5941 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt
     6001 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     6061 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     6121 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     6181 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     6241 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta
     6301 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     6361 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     6421 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     6481 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     6541 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     6601 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     6661 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     6721 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     6781 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     6841 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     6901 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     6961 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     7021 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     7081 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     7141 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggt
//
