LOCUS       sequence-404738-        5824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transferrin receptor (TfR)-based construct to display superfolder 
            green fluorescent protein (sfGFP) linked to SnoopTag2 on the surface
            of mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5824)
  AUTHORS   Keeble AH, Wood DP, Howarth M
  TITLE     Design and Evolution of Enhanced Peptide-Peptide Ligation for 
            Modular Transglutaminase Assembly.
  JOURNAL   Bioconjug Chem. 2023 Jun 8. doi: 10.1021/acs.bioconjchem.3c00122.
  PUBMED    37289810
REFERENCE   2  (bases 1 to 5824)
  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: "Bioconjug 
            Chem. 2023 Jun 8. doi: 10.1021/acs.bioconjchem.3c00122."
COMMENT     Sequence Label: pENTR4 TfR-sfGFP-SnoopTag2
FEATURES             Location/Qualifiers
     source          1..5824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      103..189
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      281..308
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     316..339
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    358..457
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     enhancer        980..1359
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1360..1563
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1514..1534
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    1565..1583
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1586..1604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1614..1638
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1753..2579
                     /label=CMV intron A
                     /note="human cytomegalovirus intron A (Chapman et al., 
                     1991)"
     CDS             2897..3610
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             3623..3658
                     /label=SnoopTag2
     promoter        complement(3723..3741)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3724..3741)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3761..3778)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3767..3991
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(4053..4152)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4264..4283)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4275..5084
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4342..4361)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5174..5762
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5663..5682
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg
       61 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      121 cagtcggaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      181 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg
      241 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat
      301 ggcctttttg cgtttctaca aactcttcct gttagttagt tacttaagct cgggccccaa
      361 ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga taagcaatgc
      421 ttttttataa tgccaacttt gtacaaaaaa gcaggctcca ccatgggaac ccgcgttttg
      481 agatttctgt cgccgactaa attcatgtcg cgcgatagtg gtgtttatcg ccgatagaga
      541 tggcgatatt ggaaaaatcg atatttgaaa atatggcata ttgaaaatgt cgccgatgtg
      601 agtttctgtg taactgatat cgccattttt ccaaaagtga tttttgggca tacgcgatat
      661 ctggcgatag cgcttatatc gtttacgggg gatggcgata gacgactttg gtgacttggg
      721 cgattctgtg tgtcgcaaat atcgcagttt cgatataggt gacagacgat atgaggctat
      781 atcgccgata gaggcgacat caagctggca catggccaat gcatatcgat ctatacattg
      841 aatcaatatt ggccattagc catattattc attggttata tagcataaat caatattggc
      901 tattggccat tgcatacgtt gtatccatat cataatatgt acatttatat tggctcatgt
      961 ccaacattac cgccatgttg acattgatta ttgactagtt attaatagta atcaattacg
     1021 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     1081 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     1141 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     1201 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     1261 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     1321 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     1381 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     1441 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     1501 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     1561 gctctcccta tcagtgatag agatctccct atcagtgata gagatcgtcg acgagctcgt
     1621 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
     1681 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
     1741 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcttat
     1801 gcatgctata ctgtttttgg cttggggtct atacaccccc gcttcctcat gttataggtg
     1861 atggtatagc ttagcctata ggtgtgggtt attgaccatt attgaccact cccctattgg
     1921 tgacgatact ttccattact aatccataac atggctcttt gccacaactc tctttattgg
     1981 ctatatgcca atacactgtc cttcagagac tgacacggac tctgtatttt tacaggatgg
     2041 ggtctcattt attatttaca aattcacata tacaacacca ccgtccccag tgcccgcagt
     2101 ttttattaaa cataacgtgg gatctccacg cgaatctcgg gtacgtgttc cggacatggg
     2161 ctcttctccg gtagcggcgg agcttctaca tccgagccct gctcccatgc ctccagcgac
     2221 tcatggtcgc tcggcagctc cttgctccta acagtggagg ccagacttag gcacagcacg
     2281 atgcccacca ccaccagtgt gccgcacaag gccgtggcgg tagggtatgt gtctgaaaat
     2341 gagctcgggg agcgggcttg caccgctgac gcatttggaa gacttaaggc agcggcagaa
     2401 gaagatgcag gcagctgagt tgttgtgttc tgataagagt cagaggtaac tcccgttgcg
     2461 gtgctgttaa cggtggaggg cagtgtagtc tgagcagtac tcgttgctgc cgcgcgcgcc
     2521 accagacata atagctgaca gactaacaga ctgttccttt ccatgggtct tttctgcagt
     2581 caccgtcctt gacacgaagc ttggtaccgc caccatgatg gatcaagcta gatcagcatt
     2641 ctctaacttg tttggtggag aaccattgtc atgtacccgg gccagcctgg ctcggcaagt
     2701 agatggcgat aacagtcatg tggagatgaa acttgctgta gatgaagaag aaaatgctga
     2761 caataacaca aaggccaatg tcacaaaacc aaaaaggtgt agtggaagta tctgctatgg
     2821 gactattgct gtgatcgtct ttttcttgat tggatttatg attggctact tgggctatgg
     2881 aagcggagga tccgggatgc gtaaaggcga agagctgttc actggtgtcg tccctattct
     2941 ggtggaactg gatggtgatg tcaacggtca taagttttcc gtgcgtggcg agggtgaagg
     3001 tgacgcaact aatggtaaac tgacgctgaa gttcatctgt actactggta aactgccggt
     3061 accttggccg actctggtaa cgacgctgac ttatggtgtt cagtgctttg ctcgttatcc
     3121 ggaccatatg aagcagcatg acttcttcaa gtccgccatg ccggaaggct atgtgcagga
     3181 acgcacgatt tcctttaagg atgacggcac gtacaaaacg cgtgcggaag tgaaatttga
     3241 aggcgatacc ctggtaaacc gcattgagct gaaaggcatt gactttaaag aagacggcaa
     3301 tatcctgggc cataagctgg aatacaattt taacagccac aatgtttaca tcaccgccga
     3361 taaacaaaaa aatggcatta aagcgaattt taaaattcgc cacaacgtgg aggatggcag
     3421 cgtgcagctg gctgatcact accagcaaaa cactccaatc ggtgatggtc ctgttctgct
     3481 gccagacaat cactatctga gcacgcaaag cgttctgtct aaagatccga acgagaaacg
     3541 cgatcatatg gttctgctgg agttcgtaac cgcagcgggc atcacgcatg gtatggatga
     3601 actatacaaa agcagcggcg ggaaactggg ctatattgaa ttttataaag tggaaaaagg
     3661 atctagcgga tcttaagaat tcctcgaggc ggccgctcga gcatgcatct agagggccct
     3721 attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt gccttctagt
     3781 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     3841 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     3901 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     3961 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     4021 tcgagggggg atcgatccgt cgagatatct agacccagct ttcttgtaca aagttggcat
     4081 tataagaaag cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa
     4141 aatcattatt tgccatccag ctgcagctct ggcccgtgtc tcaaaatctc tgatgttaca
     4201 ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta
     4261 atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca
     4321 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     4381 cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     4441 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     4501 ttatgcctct tccgaccatc aagcatttta tccgtactcc tggtgatgca tggttactca
     4561 ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     4621 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     4681 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     4741 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     4801 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     4861 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     4921 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     4981 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     5041 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt
     5101 ggttgtaaca ttattcagat tgggccccgt tccactgagc gtcagacccc gtagaaaaga
     5161 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5221 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5281 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5341 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5401 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5461 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5521 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5581 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5641 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5701 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5761 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5821 tgtt
//
