LOCUS       sequence-224967-        5220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repair template for the TLR reporter with 7 tet operator elements.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5220)
  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 5220)
  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-tetO
FEATURES             Location/Qualifiers
     source          1..5220
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(53..367)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    127..145
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    163..181
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    198..216
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    234..252
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    270..288
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    305..323
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    341..359
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(501..503,504..1217)
                     /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(543..564)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(804..823)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1151..1172
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1226..1282
                     /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             1283..1996
                     /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(2022..2038)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2052..2069)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2058..2282
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2376..2395)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2377..2395)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2405..2422)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2405..2421)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2414..2436)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2563..3018
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2650..2669)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2860..2881
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3044..3148
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3149..3217,3218..4009)
                     /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(3367..3386)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4180..4768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4669..4688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4922..4939
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5056..5077
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5092..5097,5098..5115,5116..5122)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5130..5146
                     /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     5135..5157
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5154..5170
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5154..5170
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5189..5209
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5191..5209
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gggtaccggg ccccccctcg aggtcgaccg gcggccgcag cgctttaatt aaggcgatct
       61 gacggttcac taaacgagct ctgcttatat aggcctccca ccgtacacgc ctacctcgac
      121 atacgttctc tatcactgat agggagtaaa ctcgacatac gttctctatc actgataggg
      181 ataaactcga catacgttct ctatcactga tagggagtaa actcgacata cgttctctat
      241 cactgatagg gagtaaactc gacatacgtt ctctatcact gatagggagt aaactcgaca
      301 tcgttctcta tcactgatag ggagtaaact cgacatacgt tctctatcac tgatagggag
      361 taaactcgac agtaccaagc tttactaggc gtgtacggtg ggaggcctat ataagcagag
      421 ctcgtttagt gaaccgtcag atcgccgcga tcgcgatcat cgatgtcgac ggtatcgata
      481 agcttgatta agccgccacc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      541 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
      601 agggcgatgc cacctacggc aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc
      661 ccgtgccctg gcccaccctc gtgaccaccc tgggctacgg cctgcagtgc ttcgcccgct
      721 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
      781 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
      841 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
      901 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcaccg
      961 ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg
     1021 gcggcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1081 tgctgcccga caaccactac ctgagctacc agtccgccct gagcaaagac cccaacgaga
     1141 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1201 acgagctgta caagtagacg cgttggccac gaacttctct ctgttaaagc aagcaggaga
     1261 tgttgaagaa aaccccgggc ctatggtgtc taagggcgaa gagctgatta aggagaacat
     1321 gcacatgaag ctgtacatgg agggcaccgt gaacaaccac cacttcaagt gcacatccga
     1381 gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg
     1441 ccctctcccc ttcgccttcg acatcctggc taccagcttc atgtacggca gcagaacctt
     1501 catcaaccac acccagggca tccccgactt ctttaagcag tccttccctg agggcttcac
     1561 atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag
     1621 cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact tcccatccaa
     1681 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc aacaccgaga tgctgtaccc
     1741 cgctgacggc ggcctggaag gcagaagcga catggccctg aagctcgtgg gcgggggcca
     1801 cctgatctgc aacttcaaga ccacatacag atccaagaaa cccgctaaga acctcaagat
     1861 gcccggcgtc tactatgtgg accacagact ggaaagaatc aaggaggccg acaaagagac
     1921 ctacgtcgag cagcacgagg tggctgtggc cagatactgc gacctcccta gcaaactggg
     1981 gcacaaactt aattgagcga tcgcacgcgt aaatgattgc agatccacta gttctagagc
     2041 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2101 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2161 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2221 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2281 ggcttctgag gcggaaagaa ccagctgggg ctcgagatcc actagttcta gcctcgaggc
     2341 tagagcggcc gccaccgcgg tggagctcca attcgcccta tagtgagtcg tattacgcgc
     2401 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     2461 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     2521 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     2581 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2641 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2701 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2761 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2821 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2881 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2941 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     3001 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3061 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3121 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     3181 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3241 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3301 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3361 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     3421 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     3481 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     3541 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3601 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3661 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     3721 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3781 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     3841 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     3901 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     3961 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     4021 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4081 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4141 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4201 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4261 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4321 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4381 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4441 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4501 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4561 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4621 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4681 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4741 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4801 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4861 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4921 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     4981 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     5041 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     5101 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     5161 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
//
