LOCUS       sequence-258698-        4777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for repair of the traffic light reporter (TLR), providing the
            venus coding region.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4777)
  AUTHORS   Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R
  TITLE     Increasing the efficiency of homology-directed repair for 
            CRISPR-Cas9-induced precise gene editing in mammalian cells.
  JOURNAL   Nat Biotechnol. 2015 Mar 24. doi: 10.1038/nbt.3198.
  PUBMED    25803306
REFERENCE   2  (bases 1 to 4777)
  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: "Nat 
            Biotechnol. 2015 Mar 24. doi: 10.1038/nbt.3198."
COMMENT     Sequence Label: pTLR repair vector
FEATURES             Location/Qualifiers
     source          1..4777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..6,7..720)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(46..67)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(307..326)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     654..675
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             729..785
                     /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             786..1499
                     /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(1525..1541)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1555..1572)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1561..1785
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1879..1898)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1880..1898)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1905..1922)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1905..1921)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1914..1936)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2062..2517)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2199..2220)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2411..2430
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2544..2648
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2649..2717,2718..3509)
                     /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(2867..2886)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3680..4268
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4169..4188
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4422..4439
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4556..4577
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4592..4597,4598..4615,4616..4622)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4630..4646
                     /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     4635..4657
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4654..4670
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4654..4670
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4691..4709
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     4737..4753
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4738..4754
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      join(4772..4777,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
       61 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      121 ggcaagctga ccctgaagct gatctgcacc accggcaagc tgcccgtgcc ctggcccacc
      181 ctcgtgacca ccctgggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag
      241 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      301 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      361 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      421 aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa gcagaagaac
      481 ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc
      541 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      601 tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      661 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtag
      721 acgcgttggc cacgaacttc tctctgttaa agcaagcagg agatgttgaa gaaaaccccg
      781 ggcctatggt gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctgtaca
      841 tggagggcac cgtgaacaac caccacttca agtgcacatc cgagggcgaa ggcaagccct
      901 acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct
      961 tcgacatcct ggctaccagc ttcatgtacg gcagcagaac cttcatcaac cacacccagg
     1021 gcatccccga cttctttaag cagtccttcc ctgagggctt cacatgggag agagtcacca
     1081 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
     1141 tcatctacaa cgtcaagatc agaggggtga acttcccatc caacggccct gtgatgcaga
     1201 agaaaacact cggctgggag gccaacaccg agatgctgta ccccgctgac ggcggcctgg
     1261 aaggcagaag cgacatggcc ctgaagctcg tgggcggggg ccacctgatc tgcaacttca
     1321 agaccacata cagatccaag aaacccgcta agaacctcaa gatgcccggc gtctactatg
     1381 tggaccacag actggaaaga atcaaggagg ccgacaaaga gacctacgtc gagcagcacg
     1441 aggtggctgt ggccagatac tgcgacctcc ctagcaaact ggggcacaaa cttaattgag
     1501 cgatcgcacg cgtaaatgat tgcagatcca ctagttctag agctcgctga tcagcctcga
     1561 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     1621 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     1681 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     1741 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
     1801 gaaccagctg gggctcgaga tccactagtt ctagcctcga ggctagagcg gccgccaccg
     1861 cggtggagct ccaattcgcc ctatagtgag tcgtattaca attcactggc cgtcgtttta
     1921 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     1981 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     2041 cgcagcctga atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     2101 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     2161 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     2221 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     2281 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     2341 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     2401 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     2461 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag
     2521 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2581 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2641 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2701 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2761 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2821 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2881 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2941 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3001 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3061 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3121 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3181 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3241 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3301 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3361 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3421 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3481 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3541 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3601 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3661 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3721 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3781 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     3841 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3901 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3961 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4021 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4081 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4141 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4201 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4261 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4321 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4381 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4441 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     4501 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     4561 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     4621 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     4681 ccaagctcga aattaaccct cactaaaggg aacaaaagct ggtaccgggc cccccctcga
     4741 ggtcgacggt atcgataagc ttgattaagc cgccacc
//
