LOCUS       sequence-368383-        6942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of soluble protein 
            sfGFP-SpyCatcher-sfCherry1-10 with a 6xHIS tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6942)
  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 6942)
  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-6xHIS
FEATURES             Location/Qualifiers
     source          1..6942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             44..52
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             58..771
                     /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             772..801
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             829..1167
                     /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(1316..1334)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             1807..1824
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1877..1895)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1891..1938
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1975..2430
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2062..2081)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2272..2293
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2522..3337)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2522..3337)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3245..3264
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3329..3348
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3459..4047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3948..3967
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4201..4218
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4233..4372
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4298..4317)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4458..4480
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4474..4665)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5474..6556)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6518..6537
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6557..6634)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6840..6859
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactatagga tagtaagtct tgagactaga aagtaaggag gttttttatg
       61 cgtaaaggcg aagagctgtt cactggtgtc gtccctattc tggtggaact ggatggtgat
      121 gtcaacggtc ataagttttc cgtgcgtggc gagggtgaag gtgacgcaac taatggtaaa
      181 ctgacgctga agttcatctg tactactggt aaactgccgg taccttggcc gactctggta
      241 acgacgctga cttatggtgt tcagtgcttt gctcgttatc cggaccatat gaagcagcat
      301 gacttcttca agtccgccat gccggaaggc tatgtgcagg aacgcacgat ttcctttaag
      361 gatgacggca cgtacaaaac gcgtgcggaa gtgaaatttg aaggcgatac cctggtaaac
      421 cgcattgagc tgaaaggcat tgactttaaa gaagacggca atatcctggg ccataagctg
      481 gaatacaatt ttaacagcca caatgtttac atcaccgccg ataaacaaaa aaatggcatt
      541 aaagcgaatt ttaaaattcg ccacaacgtg gaggatggca gcgtgcagct ggctgatcac
      601 taccagcaaa acactccaat cggtgatggt cctgttctgc tgccagacaa tcactatctg
      661 agcacgcaaa gcgttctgtc taaagatccg aacgagaaac gcgatcatat ggttctgctg
      721 gagttcgtaa ccgcagcggg catcacgcat ggtatggatg aactatacaa agaacaaaaa
      781 ctcatctcag aagaggatct gggttccggt ggctcgggtg gatccatggt gaccacactg
      841 tccggactgt ctggagagca gggaccatcc ggcgacatga ccacagagga ggattctgcc
      901 acacacatca agttcagcaa gagggacgag gacggaagag agctggcagg agcaaccatg
      961 gagctgaggg atagctccgg caagaccatc agcacatgga tctccgacgg ccacgtgaag
     1021 gatttctacc tgtatcccgg caagtacacc tttgtggaga cagcagcacc agacggatat
     1081 gaggtggcaa cccctatcga gtttacagtg aacgaggacg gacaggtgac cgtggatgga
     1141 gaggcaacag agggcgatgc acacaccgga tctagcggat ctatggagga ggacaacatg
     1201 gccatcatca aggagttcat gagattcaag gtgcacatgg agggcagcgt gaacggccac
     1261 gagttcgaga tcgagggcga gggcgagggc cacccctacg agggcaccca gaccgccaag
     1321 ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgag cccccagttc
     1381 atgtacggca gcaaggccta cgtgaagcac cccgccgaca tccccgacta cctgaagctg
     1441 agcttccccg agggcttcac ctgggagaga gtgatgaact tcgaggacgg cggcgtggtg
     1501 accgtgaccc aggacagcag cctgcaggac ggccagttca tctacaaggt gaagctgctg
     1561 ggcatcaact tccccagcga cggccccgtg atgcagaaga agaccatggg ctgggaggcc
     1621 agcaccgaga gaatgtaccc cgaggacggc gccctgaagg gcgagatcaa ccagagactg
     1681 aagctgaagg acggcggcca ctacgacgcc gaggtgaaga ccacctacaa ggccaagaag
     1741 cccgtgcagc tgcccggcgc ctacaacgtg gacatcaagc tggacatcac cagccacaac
     1801 gaggaccacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     1861 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1921 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     1981 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2041 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2101 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2161 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2221 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2281 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2341 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2401 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     2461 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     2521 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     2581 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     2641 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     2701 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     2761 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     2821 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     2881 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     2941 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     3001 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     3061 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     3121 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     3181 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     3241 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     3301 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     3361 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3421 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3481 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3541 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3601 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3661 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3721 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3781 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3841 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3901 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3961 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4021 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4081 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4141 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4201 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     4261 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     4321 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     4381 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4441 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4501 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     4561 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     4621 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     4681 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     4741 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     4801 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     4861 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     4921 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     4981 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     5041 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     5101 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     5161 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     5221 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     5281 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     5341 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     5401 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     5461 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5521 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     5581 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     5641 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     5701 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     5761 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     5821 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     5881 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     5941 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     6001 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     6061 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     6121 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     6181 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     6241 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     6301 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     6361 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     6421 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     6481 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     6541 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     6601 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     6661 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     6721 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     6781 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     6841 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     6901 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa at
//
