LOCUS       sequence-364231-        7214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct for expression of tagRFP-T.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7214)
  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 7214)
  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-tagRFP
FEATURES             Location/Qualifiers
     source          1..7214
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(114..133)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      927..1515
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1416..1435
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1669..1686
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1803..1824
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1839..1844,1845..1862,1863..1869)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1877..1893
                     /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     1882..1904
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1901..1917
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1901..1917
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1936..1956
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1938..1956
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1984..2210
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             2211..2391
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2438..2563
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3056..3289
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3474..3518
                     /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        3816..4064
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3816..3836
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3987..4006
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    4127..4244
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4293..4803
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4722..4740
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4831..5562
                     /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..7214,1..756)
                     /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 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
       61 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
      121 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
      181 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
      241 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
      301 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
      361 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
      421 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
      481 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
      541 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
      601 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
      661 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
      721 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
      781 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
      841 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
      901 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
      961 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     1021 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     1081 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     1141 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     1201 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     1261 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     1321 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     1381 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     1441 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     1501 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     1561 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     1621 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     1681 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     1741 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     1801 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     1861 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     1921 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct
     1981 taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac
     2041 atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg
     2101 atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga accactgaat
     2161 tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct
     2221 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     2281 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     2341 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
     2401 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
     2461 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
     2521 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
     2581 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
     2641 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
     2701 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
     2761 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
     2821 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
     2881 taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg
     2941 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     3001 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     3061 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     3121 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     3181 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     3241 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     3301 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     3361 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     3421 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     3481 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     3541 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     3601 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     3661 ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag
     3721 gtggagagag agacagagac agatccattc gattagtgaa cggatctcga cggtatcgat
     3781 cacgagacta gcctcgagcg gccgccccct tcaccgaggg cctatttccc atgattcctt
     3841 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     3901 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     3961 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     4021 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggtccg caggtatgca
     4081 cgcgtgaatt ctcgacctcg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     4141 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     4201 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     4261 acagcagaga tccactttgg ccgcggctcg agggggttgg ggttgcgcct tttccaaggc
     4321 agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa acgcagcggc
     4381 gccgaccctg ggtctcgcac attcttcacg tccgttcgca gcgtcacccg gatcttcgcc
     4441 gctacccttg tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc
     4501 ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg cacgtctcac tagtaccctc
     4561 gcagacggac agcgccaggg agcaatggca gcgcgccgac cgcgatgggc tgtggccaat
     4621 agcggctgct cagcagggcg cgccgagagc agcggccggg aaggggcggt gcgggaggcg
     4681 gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag
     4741 cctccggagc gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga cctctctccc
     4801 cagggggatc cgacgtcacc ggagcttacc atggtgtcta agggcgaaga gctgattaag
     4861 gagaacatgc acatgaagct gtacatggag ggcaccgtga acaaccacca cttcaagtgc
     4921 acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc
     4981 gagggcggcc ctctcccctt cgccttcgac atcctggcta ccagcttcat gtacggcagc
     5041 agaaccttca tcaaccacac ccagggcatc cccgatttct ttaagcagtc cttccctgag
     5101 ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag
     5161 gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc
     5221 ccatccaacg gccctgtgat gcagaagaaa acactcggct gggaggccaa caccgagacc
     5281 ctgtaccccg ctgacggcgg cctggaaggc agaaccgaca tggccctgaa gctcgtgggc
     5341 gggggccacc tgatctgcaa cttcaagacc acatacagat ccaagaaacc cgctaagaac
     5401 ctcaagatgc ccggcgtcta ctatgtggac cacagactgg aaagaatcaa ggaggccgac
     5461 aaagagacct acgtcgagca gcacgaggtg gctgtggcca gatactgcga cctccctagc
     5521 aaactggggc acaaacttaa tggcatggac gagctgtaca agtaacgtac 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 tgaa
//
