LOCUS       sequence-228917-        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        287..693
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      708..717
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(759..780)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1041..1060)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1388..1409
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1466..1519
                     /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             1520..2227
                     /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(1662..1680)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1930..1949
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        2254..2465
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2481..3080
                     /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(2481..2500)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2977..2997
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3094..3682
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3147..3167)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3565..3576)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(3758..3791)
                     /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(3996..4013)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4002..4226
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4299..4316)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4470..5058)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4550..4569)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5229..6020,6021..6089))
                     /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     5852..5871
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6090..6194)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        6228..6454
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6455..6635
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6682..6807
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7299..7532
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7717..7761
                     /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    8028..8145
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 tcctcgcccc gcctccccct tccctcccct tggggaccac cgggcgccac gccgcgaacg
       61 gtaagtgccg cggtcgtcgg cgcctccgcc ctccccctag ggccccaatt cccagcgggc
      121 gcggcgcgcg gccccgcgcg cagctcccgg ctccctcccc cttcggatgt ggcttgagct
      181 gtaggcgcgg agtcctgcag ccccgataaa ataaaagatt ttatttagtc tccagaaaaa
      241 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gctgcagtaa cgccattttg
      301 caaggcatgg aaaaatacca aaccaagaat agagaagttc agatcaaggg cgggtacatg
      361 aaaatagcta acgttgggcc aaacaggata tctgcggtga gcagtttcgg ccccggcccg
      421 gggccaagaa cagatggtca ccgcagtttc ggccccggcc cgaggccaag aacagatggt
      481 ccccagatat ggcccaaccc tcagcagttt cttaagaccc atcagatgtt tccaggctcc
      541 cccaaggacc tgaaatgacc ctgcgcctta tttgaattaa ccaatcagcc tgcttctcgc
      601 ttctgttcgc gcgcttctgc ttcccgagct ctataaaaga gctcacaacc cctcactcgg
      661 cgcgccagtc ctccgacaga ctgagtcgcc cggatcgatc ctcgagcgcc accatggtga
      721 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
      781 taaacggcca caagttcagc gtgtccggct ttggcgagac aaatcacctg cctcgtggaa
      841 tacggtaaac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      901 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      961 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     1021 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1081 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1141 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1201 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1261 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1321 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     1381 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1441 agctgtacaa gtaactggat ccggtgaggg cagaggaagt cttctaacat gcggtgacgt
     1501 ggaggagaat ccgggccctg tgagcaaggg cgaggaggat aactccgcca tcatcaagga
     1561 gttcctgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
     1621 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     1681 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
     1741 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
     1801 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
     1861 ctcctctctg caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
     1921 ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat
     1981 gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg
     2041 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc
     2101 cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat
     2161 cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta
     2221 caagtgaatg cagaggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
     2281 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
     2341 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
     2401 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
     2461 cacaggatcc tcgagccacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     2521 acgacgtccc ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     2581 gccacaccgt cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     2641 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     2701 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     2761 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     2821 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc
     2881 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     2941 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     3001 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     3061 cccgcaagcc cggtgcctga atctagtgtc gacaatcaac ctctggatta caaaatttgt
     3121 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     3181 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     3241 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     3301 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     3361 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     3421 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     3481 tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc
     3541 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     3601 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     3661 tccctttggg ccgcctcccc gcctggaatt cggtaccttt ttaaaagaaa aggggggact
     3721 ggaagggcta attcactccc aacgaagaca agatatcata acttcgtata gcatacatta
     3781 tacgaagtta taatttattt gtgaaatttg tgatgctatt gctttatttg taaccatatg
     3841 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattgcttt ttgcttgtac
     3901 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     3961 actgcttaag cctcaataaa gcttgcctcg accagcctcg actgtgcctt ctagttgcca
     4021 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     4081 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     4141 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     4201 tgctggggat gcggtgggct ctatggcctg cagctgcatt aatgaatcgg ccaacgcgcg
     4261 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4321 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4381 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4441 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4501 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4561 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4621 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4681 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4741 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4801 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4861 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4921 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4981 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5041 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5101 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5161 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5221 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5281 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5341 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5401 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5461 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5521 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5581 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5641 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5701 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5761 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5821 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5881 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5941 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6001 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6061 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6121 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6181 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcaatgt agtcttatgc
     6241 aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga
     6301 gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg
     6361 aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga
     6421 tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg
     6481 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     6541 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     6601 cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa
     6661 gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg
     6721 gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag
     6781 aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg
     6841 gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg
     6901 gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc
     6961 tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga
     7021 tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac
     7081 accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag
     7141 caagcggccc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa
     7201 ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag
     7261 agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc
     7321 ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga
     7381 caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa
     7441 cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct
     7501 gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc
     7561 atttgcacca ctgctgtgcc ttggaatgct agttggagta atgaatctct ggaacagatt
     7621 tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata
     7681 cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa
     7741 ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata
     7801 aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt
     7861 tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca
     7921 accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga
     7981 gacagatcca ttcgattagt gaacggatct cgacggtatc ggttaacttt taaaagaaaa
     8041 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     8101 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     8161 gacagcagag atccagtttg gatcgataag cttgatatcg aattcgggag gtggtccctg
     8221 cagttacgcc aatgataacc cccgccagaa aaatcttagt agccttccct ttttgttttc
     8281 cgtgccccaa ctcggcggat tgactcggcc ccttccggaa acacccgaat caacttctag
     8341 tcaaattatt gttcacgccg caatgaccca cccctggccc gcgtctgtgg aactgacccc
     8401 tggtgtacag gagagttcgc tgctgaaagt ggtcccaaag gggtactagt ttttaagctc
     8461 ccaactcccc ctcccccagc gtctggagga ttccacaccc tcgcaccgca ggggcgagga
     8521 agtgggcgga gtccggtttt ggcgccagcc gctgaggctg ccaagcagaa aagccaccgc
     8581 tgaggagact ccggtcactg
//
