LOCUS       sequence-368384-        5577 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free expression of transmembrane protein 
            TfR-sfGFP-SpyCatcher-sfCherry1-10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5577)
  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 5577)
  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: InterCatch-GFP
FEATURES             Location/Qualifiers
     source          1..5577
                     /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             818..1531
                     /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             1532..1561
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1589..1927
                     /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(2076..2094)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2640..2658)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2654..2701
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2730..2747)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2901..3489)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2981..3000)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3660..4451,4452..4520))
                     /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     4283..4302
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4521..4625)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4693..4711
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4749..4771)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4871..4890
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4959..5414)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5096..5117)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5308..5327
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5540..5562
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5555..5571
                     /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 atatgatgat
      541 ggatcaagct agatcagcat tctctaactt gtttggtgga gaaccattgt catgtacccg
      601 ggccagcctg gctcggcaag tagatggcga taacagtcat gtggagatga aacttgctgt
      661 agatgaagaa gaaaatgctg acaataacac aaaggccaat gtcacaaaac caaaaaggtg
      721 tagtggaagt atctgctatg ggactattgc tgtgatcgtc tttttcttga ttggatttat
      781 gattggctac ttgggctatg gaagcggagg atccgggatg cgtaaaggcg aagagctgtt
      841 cactggtgtc gtccctattc tggtggaact ggatggtgat gtcaacggtc ataagttttc
      901 cgtgcgtggc gagggtgaag gtgacgcaac taatggtaaa ctgacgctga agttcatctg
      961 tactactggt aaactgccgg taccttggcc gactctggta acgacgctga cttatggtgt
     1021 tcagtgcttt gctcgttatc cggaccatat gaagcagcat gacttcttca agtccgccat
     1081 gccggaaggc tatgtgcagg aacgcacgat ttcctttaag gatgacggca cgtacaaaac
     1141 gcgtgcggaa gtgaaatttg aaggcgatac cctggtaaac cgcattgagc tgaaaggcat
     1201 tgactttaaa gaagacggca atatcctggg ccataagctg gaatacaatt ttaacagcca
     1261 caatgtttac atcaccgccg ataaacaaaa aaatggcatt aaagcgaatt ttaaaattcg
     1321 ccacaacgtg gaggatggca gcgtgcagct ggctgatcac taccagcaaa acactccaat
     1381 cggtgatggt cctgttctgc tgccagacaa tcactatctg agcacgcaaa gcgttctgtc
     1441 taaagatccg aacgagaaac gcgatcatat ggttctgctg gagttcgtaa ccgcagcggg
     1501 catcacgcat ggtatggatg aactatacaa agaacaaaaa ctcatctcag aagaggatct
     1561 gggttccggt ggctcgggtg gatccatggt gaccacactg tccggactgt ctggagagca
     1621 gggaccatcc ggcgacatga ccacagagga ggattctgcc acacacatca agttcagcaa
     1681 gagggacgag gacggaagag agctggcagg agcaaccatg gagctgaggg atagctccgg
     1741 caagaccatc agcacatgga tctccgacgg ccacgtgaag gatttctacc tgtatcccgg
     1801 caagtacacc tttgtggaga cagcagcacc agacggatat gaggtggcaa cccctatcga
     1861 gtttacagtg aacgaggacg gacaggtgac cgtggatgga gaggcaacag agggcgatgc
     1921 acacaccgga tctagcggat ctatggagga ggacaacatg gccatcatca aggagttcat
     1981 gagattcaag gtgcacatgg agggcagcgt gaacggccac gagttcgaga tcgagggcga
     2041 gggcgagggc cacccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg
     2101 ccccctgccc ttcgcctggg acatcctgag cccccagttc atgtacggca gcaaggccta
     2161 cgtgaagcac cccgccgaca tccccgacta cctgaagctg agcttccccg agggcttcac
     2221 ctgggagaga gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggacagcag
     2281 cctgcaggac ggccagttca tctacaaggt gaagctgctg ggcatcaact tccccagcga
     2341 cggccccgtg atgcagaaga agaccatggg ctgggaggcc agcaccgaga gaatgtaccc
     2401 cgaggacggc gccctgaagg gcgagatcaa ccagagactg aagctgaagg acggcggcca
     2461 ctacgacgcc gaggtgaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     2521 ctacaacgtg gacatcaagc tggacatcac cagccacaac gaggactagc accaccacca
     2581 ctaataaaga tctgactgaa aaaaaaaaaa aaaaaaaaaa aaaaagttta aacactagtc
     2641 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     2701 gctgaaagga ggaactatat ccgggcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2761 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2821 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2881 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2941 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     3001 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3061 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     3121 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3181 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3241 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3301 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     3361 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3421 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3481 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3541 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3601 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3661 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3721 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3781 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3841 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3901 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3961 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     4021 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4081 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     4141 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     4201 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     4261 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     4321 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     4381 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     4441 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     4501 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4561 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4621 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     4681 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     4741 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     4801 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     4861 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatatg cggtgtgaaa
     4921 taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg ttaatatttt
     4981 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     5041 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     5101 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     5161 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     5221 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     5281 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     5341 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     5401 gctacagggc gcgtccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     5461 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     5521 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattg
//
