LOCUS       sequence-233153-        5216 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 5216)
  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 5216)
  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..5216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(126..128,129..842)
                     /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(168..189)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(429..448)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     776..797
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             851..907
                     /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             908..1621
                     /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(1647..1663)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1677..1694)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1683..1907
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2001..2020)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2002..2020)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2027..2044)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2027..2043)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2036..2058)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2184..2639)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2321..2342)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2533..2552
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2666..2770
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2771..2839,2840..3631)
                     /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(2989..3008)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3802..4390
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4291..4310
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4544..4561
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4678..4699
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4714..4719,4720..4737,4738..4744)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4752..4768
                     /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     4757..4779
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4776..4792
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4776..4792
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4813..4831
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(4894..5208)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    4968..4986
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5004..5022
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5039..5057
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5075..5093
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5111..5129
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5146..5164
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5182..5200
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 caagctttac taggcgtgta cggtgggagg cctatataag cagagctcgt ttagtgaacc
       61 gtcagatcgc cgcgatcgcg atcatcgatg tcgacggtat cgataagctt gattaagccg
      121 ccaccgtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      181 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
      241 acggcaagct gaccctgaag ctgatctgca ccaccggcaa gctgcccgtg ccctggccca
      301 ccctcgtgac caccctgggc tacggcctgc agtgcttcgc ccgctacccc gaccacatga
      361 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
      421 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
      481 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
      541 acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga
      601 acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg
      661 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
      721 actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
      781 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
      841 agacgcgttg gccacgaact tctctctgtt aaagcaagca ggagatgttg aagaaaaccc
      901 cgggcctatg gtgtctaagg gcgaagagct gattaaggag aacatgcaca tgaagctgta
      961 catggagggc accgtgaaca accaccactt caagtgcaca tccgagggcg aaggcaagcc
     1021 ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc
     1081 cttcgacatc ctggctacca gcttcatgta cggcagcaga accttcatca accacaccca
     1141 gggcatcccc gacttcttta agcagtcctt ccctgagggc ttcacatggg agagagtcac
     1201 cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg
     1261 cctcatctac aacgtcaaga tcagaggggt gaacttccca tccaacggcc ctgtgatgca
     1321 gaagaaaaca ctcggctggg aggccaacac cgagatgctg taccccgctg acggcggcct
     1381 ggaaggcaga agcgacatgg ccctgaagct cgtgggcggg ggccacctga tctgcaactt
     1441 caagaccaca tacagatcca agaaacccgc taagaacctc aagatgcccg gcgtctacta
     1501 tgtggaccac agactggaaa gaatcaagga ggccgacaaa gagacctacg tcgagcagca
     1561 cgaggtggct gtggccagat actgcgacct ccctagcaaa ctggggcaca aacttaattg
     1621 agcgatcgca cgcgtaaatg attgcagatc cactagttct agagctcgct gatcagcctc
     1681 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     1741 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     1801 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     1861 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     1921 aagaaccagc tggggctcga gatccactag ttctagcctc gaggctagag cggccgccac
     1981 cgcggtggag ctccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt
     2041 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     2101 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     2161 tgcgcagcct gaatggcgaa tggaaattgt aagcgttaat attttgttaa aattcgcgtt
     2221 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     2281 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     2341 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     2401 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     2461 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     2521 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     2581 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     2641 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2701 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2761 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2821 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2881 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2941 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3001 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3061 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3121 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3181 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3241 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3301 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3361 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3421 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3481 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3541 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3601 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3661 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3721 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3781 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3841 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3901 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3961 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4021 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4081 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4141 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4201 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4261 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4321 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4381 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4441 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4501 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4561 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     4621 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     4681 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     4741 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     4801 cgccaagctc gaaattaacc ctcactaaag ggaacaaaag ctggtaccgg gccccccctc
     4861 gaggtcgacc ggcggccgca gcgctttaat taaggcgatc tgacggttca ctaaacgagc
     4921 tctgcttata taggcctccc accgtacacg cctacctcga catacgttct ctatcactga
     4981 tagggagtaa actcgacata cgttctctat cactgatagg gataaactcg acatacgttc
     5041 tctatcactg atagggagta aactcgacat acgttctcta tcactgatag ggagtaaact
     5101 cgacatacgt tctctatcac tgatagggag taaactcgac atcgttctct atcactgata
     5161 gggagtaaac tcgacatacg ttctctatca ctgataggga gtaaactcga cagtac
//
