LOCUS       sequence-233146-        4774 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 4774)
  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 4774)
  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..4774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(227..246)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     574..595
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             649..705
                     /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             706..1419
                     /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(1445..1461)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1475..1492)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1481..1705
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1799..1818)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1800..1818)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1825..1842)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1825..1841)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1834..1856)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(1982..2437)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2119..2140)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2331..2350
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2464..2568
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2569..2637,2638..3429)
                     /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(2787..2806)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3600..4188
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4089..4108
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4342..4359
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4476..4497
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4512..4517,4518..4535,4536..4542)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4550..4566
                     /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     4555..4577
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4574..4590
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4574..4590
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4611..4629
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     4657..4673
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4658..4674
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(4698..4700,4701..4774,1..640)
                     /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(4740..4761)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagct
       61 gatctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgggcta
      121 cggcctgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc
      181 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
      241 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
      301 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
      361 cagccacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg ccaacttcaa
      421 gatccgccac aacatcgagg acggcggcgt gcagctcgcc gaccactacc agcagaacac
      481 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccgc
      541 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
      601 cgccgggatc actctcggca tggacgagct gtacaagtag acgcgttggc cacgaacttc
      661 tctctgttaa agcaagcagg agatgttgaa gaaaaccccg ggcctatggt gtctaagggc
      721 gaagagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac cgtgaacaac
      781 caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg
      841 agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct ggctaccagc
      901 ttcatgtacg gcagcagaac cttcatcaac cacacccagg gcatccccga cttctttaag
      961 cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga cgggggcgtg
     1021 ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaagatc
     1081 agaggggtga acttcccatc caacggccct gtgatgcaga agaaaacact cggctgggag
     1141 gccaacaccg agatgctgta ccccgctgac ggcggcctgg aaggcagaag cgacatggcc
     1201 ctgaagctcg tgggcggggg ccacctgatc tgcaacttca agaccacata cagatccaag
     1261 aaacccgcta agaacctcaa gatgcccggc gtctactatg tggaccacag actggaaaga
     1321 atcaaggagg ccgacaaaga gacctacgtc gagcagcacg aggtggctgt ggccagatac
     1381 tgcgacctcc ctagcaaact ggggcacaaa cttaattgag cgatcgcacg cgtaaatgat
     1441 tgcagatcca ctagttctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     1501 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     1561 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     1621 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     1681 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgaga
     1741 tccactagtt ctagcctcga ggctagagcg gccgccaccg cggtggagct ccaattcgcc
     1801 ctatagtgag tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     1861 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     1921 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     1981 gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     2041 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     2101 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     2161 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     2221 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     2281 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     2341 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     2401 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
     2461 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2521 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2581 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2641 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2701 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     2761 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     2821 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     2881 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     2941 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     3001 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     3061 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     3121 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     3181 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     3241 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     3301 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     3361 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     3421 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3481 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3541 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3601 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3661 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3721 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     3781 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3841 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3901 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3961 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4021 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4081 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4141 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4201 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4261 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4321 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     4381 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     4441 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     4501 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     4561 taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcga aattaaccct
     4621 cactaaaggg aacaaaagct ggtaccgggc cccccctcga ggtcgacggt atcgataagc
     4681 ttgattaagc cgccaccgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     4741 tggtcgagct ggacggcgac gtaaacggcc acaa
//
