LOCUS       sequence-224966-        4778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repair template for the TLR reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4778)
  AUTHORS   Bashir S, Dang T, Rossius J, Wolf J, Kuhn R
  TITLE     Enhancement of CRISPR-Cas9 induced precise gene editing by targeting
            histone H2A-K15 ubiquitination.
  JOURNAL   BMC Biotechnol. 2020 Oct 23;20(1):57. doi: 
            10.1186/s12896-020-00650-x.
  PUBMED    33097066
REFERENCE   2  (bases 1 to 4778)
  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: "BMC 
            Biotechnol."; date: "2020-10-23"; volume: "20(1):57. doi"; pages: " 
            10.1186/s12896-020-00650-x"
COMMENT     Sequence Label: pTLR-donor
FEATURES             Location/Qualifiers
     source          1..4778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..34
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     19..35
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(59..61,62..775)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(101..122)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(362..381)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     709..730
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             784..840
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             841..1554
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     primer_bind     complement(1580..1596)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1610..1627)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1616..1840
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1934..1953)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1935..1953)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1963..1980)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1963..1979)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1972..1994)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2121..2576
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2208..2227)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2418..2439
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2602..2706
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2707..2775,2776..3567)
                     /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     complement(2925..2944)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3738..4326
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4227..4246
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4480..4497
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4614..4635
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4650..4655,4656..4673,4674..4680)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4688..4704
                     /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     4693..4715
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4712..4728
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4712..4728
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4747..4767
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4749..4767
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gggtaccggg ccccccctcg aggtcgacgg tatcgataag cttgattaag ccgccaccgt
       61 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
      121 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
      181 gctgaccctg aagctgatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
      241 gaccaccctg ggctacggcc tgcagtgctt cgcccgctac cccgaccaca tgaagcagca
      301 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
      361 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
      421 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
      481 ggagtacaac tacaacagcc acaacgtcta tatcaccgcc gacaagcaga agaacggcat
      541 caaggccaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc tcgccgacca
      601 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
      661 gagctaccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
      721 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtagacgcg
      781 ttggccacga acttctctct gttaaagcaa gcaggagatg ttgaagaaaa ccccgggcct
      841 atggtgtcta agggcgaaga gctgattaag gagaacatgc acatgaagct gtacatggag
      901 ggcaccgtga acaaccacca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag
      961 ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac
     1021 atcctggcta ccagcttcat gtacggcagc agaaccttca tcaaccacac ccagggcatc
     1081 cccgacttct ttaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac
     1141 gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc
     1201 tacaacgtca agatcagagg ggtgaacttc ccatccaacg gccctgtgat gcagaagaaa
     1261 acactcggct gggaggccaa caccgagatg ctgtaccccg ctgacggcgg cctggaaggc
     1321 agaagcgaca tggccctgaa gctcgtgggc gggggccacc tgatctgcaa cttcaagacc
     1381 acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcgtcta ctatgtggac
     1441 cacagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca gcacgaggtg
     1501 gctgtggcca gatactgcga cctccctagc aaactggggc acaaacttaa ttgagcgatc
     1561 gcacgcgtaa atgattgcag atccactagt tctagagctc gctgatcagc ctcgactgtg
     1621 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1681 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1741 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     1801 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     1861 agctggggct cgagatccac tagttctagc ctcgaggcta gagcggccgc caccgcggtg
     1921 gagctccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca
     1981 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     2041 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     2101 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2161 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2221 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2281 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     2341 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     2401 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     2461 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     2521 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     2581 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2641 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2701 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     2761 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2821 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2881 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     2941 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3001 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3061 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     3121 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     3181 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     3241 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     3301 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     3361 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     3421 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     3481 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     3541 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     3601 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     3661 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3721 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3781 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3841 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3901 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3961 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4021 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4081 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4141 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4201 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4261 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4321 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4381 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4441 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4501 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     4561 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     4621 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     4681 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     4741 aagcgcgcaa ttaaccctca ctaaagggaa caaaagct
//
