LOCUS       sequence-221574-        8600 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector encoding Traffic light reporter 2.0. Alternative 
            name: pCVL-UCOE-SFFV-TLR-MCV1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8600)
  AUTHORS   Iyer S, Mir A, Vega-Badillo J, Roscoe BP, Ibraheim R, Zhu LJ, Lee J,
            Liu P, Luk K, Mintzer E, Guo D, Soares de Brito J, Emerson CP Jr, 
            Zamore PD, Sontheimer EJ, Wolfe SA
  TITLE     Efficient Homology-Directed Repair with Circular Single-Stranded DNA
            Donors.
  JOURNAL   CRISPR J. 2022 Oct;5(5):685-701. doi: 10.1089/crispr.2022.0058. Epub
            2022 Sep 7.
  PUBMED    36070530
REFERENCE   2  (bases 1 to 8600)
  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"; doi: 
            "10.1089/crispr.2022.0058"; journalName: "CRISPR J"; date: 
            "2022-10"; volume: "5"; issue: "5"; pages: "685-701"
COMMENT     Sequence Label: pCVL-UCOE-SFFV-TLR-MCV1
FEATURES             Location/Qualifiers
     source          1..8600
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..235
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             236..416
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    463..588
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1080..1313
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1498..1542
                     /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    1809..1926
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2668..3074
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      3089..3098
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3140..3161)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3422..3441)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3769..3790
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3847..3900
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3901..4608
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNSAIIKEFLRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(4043..4061)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4311..4330
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        4635..4846
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             4862..5461
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4862..4881)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5358..5378
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    5475..6063
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5528..5548)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5946..5957)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(6139..6172)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(6377..6394)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6383..6607
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6680..6697)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6851..7439)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6931..6950)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7610..8401,8402..8470))
                     /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"
     primer_bind     8233..8252
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8471..8575)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
       61 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
      121 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
      181 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc
      241 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      301 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      361 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      421 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
      481 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
      541 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
      601 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
      661 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
      721 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
      781 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
      841 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
      901 aaaagtaaga ccaccgcaca gcaagcggcc ctgatcttca gacctggagg aggagatatg
      961 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1021 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1081 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1141 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1201 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1261 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1321 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1381 aatgaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     1441 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     1501 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     1561 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     1621 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     1681 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     1741 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat
     1801 cggttaactt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     1861 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     1921 aattttcggg tttattacag ggacagcaga gatccagttt ggatcgataa gcttgatatc
     1981 gaattcggga ggtggtccct gcagttacgc caatgataac ccccgccaga aaaatcttag
     2041 tagccttccc tttttgtttt ccgtgcccca actcggcgga ttgactcggc cccttccgga
     2101 aacacccgaa tcaacttcta gtcaaattat tgttcacgcc gcaatgaccc acccctggcc
     2161 cgcgtctgtg gaactgaccc ctggtgtaca ggagagttcg ctgctgaaag tggtcccaaa
     2221 ggggtactag tttttaagct cccaactccc cctcccccag cgtctggagg attccacacc
     2281 ctcgcaccgc aggggcgagg aagtgggcgg agtccggttt tggcgccagc cgctgaggct
     2341 gccaagcaga aaagccaccg ctgaggagac tccggtcact gtcctcgccc cgcctccccc
     2401 ttccctcccc ttggggacca ccgggcgcca cgccgcgaac ggtaagtgcc gcggtcgtcg
     2461 gcgcctccgc cctcccccta gggccccaat tcccagcggg cgcggcgcgc ggccccgcgc
     2521 gcagctcccg gctccctccc ccttcggatg tggcttgagc tgtaggcgcg gagtcctgca
     2581 gccccgataa aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc
     2641 acctgtaggt ttggcaagct agctgcagta acgccatttt gcaaggcatg gaaaaatacc
     2701 aaaccaagaa tagagaagtt cagatcaagg gcgggtacat gaaaatagct aacgttgggc
     2761 caaacaggat atctgcggtg agcagtttcg gccccggccc ggggccaaga acagatggtc
     2821 accgcagttt cggccccggc ccgaggccaa gaacagatgg tccccagata tggcccaacc
     2881 ctcagcagtt tcttaagacc catcagatgt ttccaggctc ccccaaggac ctgaaatgac
     2941 cctgcgcctt atttgaatta accaatcagc ctgcttctcg cttctgttcg cgcgcttctg
     3001 cttcccgagc tctataaaag agctcacaac ccctcactcg gcgcgccagt cctccgacag
     3061 actgagtcgc ccggatcgat cctcgagcgc caccatggtg agcaagggcg aggagctgtt
     3121 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     3181 cgtgtccggc tttggcgaga caaatcacct gcctcgtgga atacggtaaa cctacggcaa
     3241 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     3301 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     3361 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     3421 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     3481 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     3541 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     3601 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     3661 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     3721 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     3781 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaactgga
     3841 tccggtgagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tccgggccct
     3901 gtgagcaagg gcgaggagga taactccgcc atcatcaagg agttcctgcg cttcaaggtg
     3961 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     4021 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     4081 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     4141 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     4201 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctctct gcaggacggc
     4261 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     4321 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     4381 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     4441 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     4501 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     4561 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtgaat gcagaggctc
     4621 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     4681 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     4741 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     4801 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggatc ctcgagccac
     4861 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc cccgggccgt
     4921 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgacccgga
     4981 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     5041 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     5101 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     5161 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     5221 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     5281 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     5341 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     5401 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     5461 aatctagtgt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     5521 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     5581 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     5641 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     5701 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     5761 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     5821 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct
     5881 ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     5941 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     6001 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     6061 cgcctggaat tcggtacctt tttaaaagaa aaggggggac tggaagggct aattcactcc
     6121 caacgaagac aagatatcat aacttcgtat agcatacatt atacgaagtt ataatttatt
     6181 tgtgaaattt gtgatgctat tgctttattt gtaaccatat gtttatttgt gaaatttgtg
     6241 atgctattgc tttatttgta accattgctt tttgcttgta ctgggtctct ctggttagac
     6301 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     6361 agcttgcctc gaccagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     6421 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     6481 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     6541 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     6601 tctatggcct gcagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6661 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     6721 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6781 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6841 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6901 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6961 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     7021 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     7081 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     7141 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     7201 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     7261 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     7321 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     7381 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     7441 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     7501 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     7561 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     7621 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     7681 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     7741 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     7801 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7861 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7921 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7981 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     8041 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     8101 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     8161 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     8221 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     8281 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     8341 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     8401 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     8461 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     8521 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     8581 ttccccgaaa agtgccacct
//
