LOCUS       sequence-67851-e        8155 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codes for the Active/Repressed TLR with the target site for I-Sce I 
            nuclease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8155)
  AUTHORS   Kuhar R, Gwiazda KS, Humbert O, Mandt T, Pangallo J, Brault M, Khan 
            I, Maizels N, Rawlings DJ, Scharenberg AM, Certo MT
  TITLE     Novel fluorescent genome editing reporters for monitoring DNA repair
            pathway utilization at endonuclease-induced breaks.
  JOURNAL   Nucleic Acids Res. 2013 Oct 10.
  PUBMED    24121685
REFERENCE   2  (bases 1 to 8155)
  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: "Nucleic 
            Acids Res. 2013 Oct 10."
COMMENT     Sequence Label: pCVL.Active/Repressed TLR (Sce)
FEATURES             Location/Qualifiers
     source          1..8155
                     /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        1955..2356
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      2383..2392
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2389..3336
                     /codon_start=1
                     /product="phytochrome-based near-infrared fluorescent 
                     protein, originally termed iRFP (Filonov et al., 2011)"
                     /label=iRFP713
                     /note="name was changed to iRFP713 (Shcherbakova and 
                     Verkhusha, 2013)"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     CDS             3343..3396
                     /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"
     primer_bind     complement(3439..3460)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3706..3725)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4053..4074
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4251..4304
                     /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             4305..5012
                     /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(4447..4465)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4715..4734
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    5024..5612
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5077..5097)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5495..5506)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(5694..5727)
                     /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(5932..5949)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5938..6162
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6235..6252)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6406..6994)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6486..6505)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7165..7956,7957..8025))
                     /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     7788..7807
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8026..8130)
                     /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 aattttatcg attacgcgtc acgtgctagc tgcagtaacg ccattttgca aggcatggaa
     1981 aaataccaaa ccaagaatag agaagttcag atcaagggcg ggtacatgaa aatagctaac
     2041 gttgggccaa acaggatatc tgcggtgagc agtttcggcc ccggcccggg gccaagaaca
     2101 gatggtcacc gcagtttcgg ccccggcccg aggccaagaa cagatggtcc ccagatatgg
     2161 cccaaccctc agcagtttct taagacccat cagatgtttc caggctcccc caaggacctg
     2221 aaatgaccct gcgccttatt tgaattaacc aatcagcctg cttctcgctt ctgttcgcgc
     2281 gcttctgctt cccgagctct ataaaagagc tcacaacccc tcactcggcg cgccagtcct
     2341 ccgacagact gagtcgatcg cttcagtcaa gatttaatta acgccaccat ggctgaagga
     2401 tccgtcgcca ggcagcctga cctcttgacc tgcgacgatg agccgatcca tatccccggt
     2461 gccatccaac cgcatggact gctgctcgcc ctcgccgccg acatgacgat cgttgccggc
     2521 agcgacaacc ttcccgaact caccggactg gcgatcggcg ccctgatcgg ccgctctgcg
     2581 gccgatgtct tcgactcgga gacgcacaac cgtctgacga tcgccttggc cgagcccggg
     2641 gcggccgtcg gagcaccgat cactgtcggc ttcacgatgc gaaaggacgc aggcttcatc
     2701 ggctcctggc atcgccatga tcagctcatc ttcctcgagc tcgagcctcc ccagcgggac
     2761 gtcgccgagc cgcaggcgtt cttccgccgc accaacagcg ccatccgccg cctgcaggcc
     2821 gccgaaacct tggaaagcgc ctgcgccgcc gcggcgcaag aggtgcggaa gattaccggc
     2881 ttcgatcggg tgatgatcta tcgcttcgcc tccgacttca gcggcgaagt gatcgcagag
     2941 gatcggtgcg ccgaggtcga gtcaaaacta ggcctgcact atcctgcctc aaccgtgccg
     3001 gcgcaggccc gtcggctcta taccatcaac ccggtacgga tcattcccga tatcaattat
     3061 cggccggtgc cggtcacccc agacctcaat ccggtcaccg ggcggccgat tgatcttagc
     3121 ttcgccatcc tgcgcagcgt ctcgcccgtc catctggaat tcatgcgcaa cataggcatg
     3181 cacggcacga tgtcgatctc gattttgcgc ggcgagcgac tgtggggatt gatcgtttgc
     3241 catcaccgaa cgccgtacta cgtcgatctc gatggccgcc aagcctgcga gctagtcgcc
     3301 caggttctgg cctggcagat cggcgtgatg gaagagtcta gagagggcag aggaagtctt
     3361 ctaacatgcg gtgacgtgga ggagaatccg ggccccgtga gcaagggcga ggagctgttc
     3421 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     3481 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     3541 accaccggca acctgcaggt attaccctgt tatccctaac tagtgtgaac cctgacctac
     3601 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     3661 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     3721 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     3781 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     3841 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     3901 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     3961 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     4021 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     4081 gccgggatca ctctcggcat ggacgagctg tacaagtaag gccggccagc cacggcttcc
     4141 cccctgaggt ggccgctcag gacgatggca ccctgcccat gagctgcgcc caggagagcg
     4201 gcatggacag gcaccccgcc gcttgcgcca gcgctaggat caacgtgggt gagggcagag
     4261 gaagtcttct aacatgcggt gacgtggagg agaatccggg ccctgtgagc aagggcgagg
     4321 aggataactc cgccatcatc aaggagttcc tgcgcttcaa ggtgcacatg gagggctccg
     4381 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     4441 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     4501 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     4561 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     4621 gcggcgtggt gaccgtgacc caggactcct ctctgcagga cggcgagttc atctacaagg
     4681 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     4741 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     4801 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     4861 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     4921 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     4981 ccaccggcgg catggacgag ctgtacaagt gaatgcatta gtcaatcaac ctctggatta
     5041 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     5101 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     5161 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     5221 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     5281 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     5341 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     5401 cgtggtgttg tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg
     5461 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     5521 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     5581 gagtcggatc tccctttggg ccgcctcccc gcctggaatt cgagctcggt acctttttaa
     5641 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat atcataactt
     5701 cgtatagcat acattatacg aagttataat ttatttgtga aatttgtgat gctattgctt
     5761 tatttgtaac catatgttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     5821 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     5881 gctaactagg gaacccactg cttaagcctc aataaagctt gcctcgacca gcctcgactg
     5941 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     6001 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     6061 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     6121 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcctgcagc tgcattaatg
     6181 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     6241 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     6301 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     6361 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     6421 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     6481 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6541 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6601 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6661 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6721 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6781 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6841 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6901 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6961 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     7021 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     7081 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     7141 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     7201 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7261 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     7321 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     7381 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     7441 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     7501 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7561 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7621 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7681 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     7741 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     7801 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     7861 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7921 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7981 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     8041 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     8101 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacct
//
