LOCUS       sequence-368385-        5294 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free expression of soluble protein 
            sfGFP-SpyCatcher-sfCherry1-10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5294)
  AUTHORS   Moghimianavval H, Patel C, Mohapatra S, Hwang SW, Kayikcioglu T, 
            Bashirzadeh Y, Liu AP, Ha T
  TITLE     Engineering Functional Membrane-Membrane Interfaces by InterSpy.
  JOURNAL   Small. 2022 May 26:e2202104. doi: 10.1002/smll.202202104.
  PUBMED    35618485
REFERENCE   2  (bases 1 to 5294)
  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: "Small. 2022
            May 26:e2202104. doi: 10.1002/smll.202202104."
COMMENT     Sequence Label: sCatch-GFP
FEATURES             Location/Qualifiers
     source          1..5294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    70..531
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(126..143)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             536..1249
                     /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             1250..1279
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1307..1645
                     /codon_start=1
                     /product="improved SpyCatcher variant that reacts with 
                     SpyTag003 at a rate approaching the diffusion limit (Keeble
                     et al., 2019)"
                     /label=SpyCatcher003
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDG
                     EATEGDAHT"
     primer_bind     complement(1794..1812)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2357..2375)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2371..2418
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2447..2464)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2618..3206)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2698..2717)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3377..4168,4169..4237))
                     /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     4000..4019
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4238..4342)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4410..4428
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4466..4488)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4588..4607
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4676..5131)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4813..4834)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5025..5044
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5257..5279
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5272..5288
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg cgaattaatt ccggttattt tccaccatat tgccgtcttt
       61 tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct
      121 ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct
      181 ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc
      241 acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc
      301 ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctcac
      361 ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc
      421 tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaacgtcta
      481 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata atatgatgcg
      541 taaaggcgaa gagctgttca ctggtgtcgt ccctattctg gtggaactgg atggtgatgt
      601 caacggtcat aagttttccg tgcgtggcga gggtgaaggt gacgcaacta atggtaaact
      661 gacgctgaag ttcatctgta ctactggtaa actgccggta ccttggccga ctctggtaac
      721 gacgctgact tatggtgttc agtgctttgc tcgttatccg gaccatatga agcagcatga
      781 cttcttcaag tccgccatgc cggaaggcta tgtgcaggaa cgcacgattt cctttaagga
      841 tgacggcacg tacaaaacgc gtgcggaagt gaaatttgaa ggcgataccc tggtaaaccg
      901 cattgagctg aaaggcattg actttaaaga agacggcaat atcctgggcc ataagctgga
      961 atacaatttt aacagccaca atgtttacat caccgccgat aaacaaaaaa atggcattaa
     1021 agcgaatttt aaaattcgcc acaacgtgga ggatggcagc gtgcagctgg ctgatcacta
     1081 ccagcaaaac actccaatcg gtgatggtcc tgttctgctg ccagacaatc actatctgag
     1141 cacgcaaagc gttctgtcta aagatccgaa cgagaaacgc gatcatatgg ttctgctgga
     1201 gttcgtaacc gcagcgggca tcacgcatgg tatggatgaa ctatacaaag aacaaaaact
     1261 catctcagaa gaggatctgg gttccggtgg ctcgggtgga tccatggtga ccacactgtc
     1321 cggactgtct ggagagcagg gaccatccgg cgacatgacc acagaggagg attctgccac
     1381 acacatcaag ttcagcaaga gggacgagga cggaagagag ctggcaggag caaccatgga
     1441 gctgagggat agctccggca agaccatcag cacatggatc tccgacggcc acgtgaagga
     1501 tttctacctg tatcccggca agtacacctt tgtggagaca gcagcaccag acggatatga
     1561 ggtggcaacc cctatcgagt ttacagtgaa cgaggacgga caggtgaccg tggatggaga
     1621 ggcaacagag ggcgatgcac acaccggatc tagcggatct atggaggagg acaacatggc
     1681 catcatcaag gagttcatga gattcaaggt gcacatggag ggcagcgtga acggccacga
     1741 gttcgagatc gagggcgagg gcgagggcca cccctacgag ggcacccaga ccgccaagct
     1801 gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgagcc cccagttcat
     1861 gtacggcagc aaggcctacg tgaagcaccc cgccgacatc cccgactacc tgaagctgag
     1921 cttccccgag ggcttcacct gggagagagt gatgaacttc gaggacggcg gcgtggtgac
     1981 cgtgacccag gacagcagcc tgcaggacgg ccagttcatc tacaaggtga agctgctggg
     2041 catcaacttc cccagcgacg gccccgtgat gcagaagaag accatgggct gggaggccag
     2101 caccgagaga atgtaccccg aggacggcgc cctgaagggc gagatcaacc agagactgaa
     2161 gctgaaggac ggcggccact acgacgccga ggtgaagacc acctacaagg ccaagaagcc
     2221 cgtgcagctg cccggcgcct acaacgtgga catcaagctg gacatcacca gccacaacga
     2281 ggactagcac caccaccact aataaagatc tgactgaaaa aaaaaaaaaa aaaaaaaaaa
     2341 aagtttaaac actagtccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     2401 gtcttgaggg gttttttgct gaaaggagga actatatccg ggcttcctcg ctcactgact
     2461 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2521 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2581 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2641 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2701 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2761 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2821 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2881 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2941 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3001 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3061 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3121 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3181 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3241 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3301 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3361 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3421 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3481 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     3541 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3601 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3661 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3721 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3781 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3841 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3901 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3961 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4021 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4081 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4141 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4201 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4261 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4321 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     4381 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     4441 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     4501 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     4561 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     4621 atatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt
     4681 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     4741 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     4801 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     4861 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     4921 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     4981 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     5041 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     5101 gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt
     5161 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     5221 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     5281 cggccagtga attg
//
