LOCUS       sequence-368390-        4405 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free expression of soluble protein tagBFP-sfCherry11-SpyTag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4405)
  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 4405)
  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: sTag-BFP
FEATURES             Location/Qualifiers
     source          1..4405
                     /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..583
                     /codon_start=1
                     /product="improved SpyTag variant that reacts with 
                     SpyCatcher003 at a rate approaching the diffusion limit 
                     (Keeble et al., 2019)"
                     /label=SpyTag003
                     /translation="RGVPHIVMVDAYKRYK"
     CDS             671..1369
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             1373..1402
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1468..1486)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1482..1529
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1558..1575)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1729..2317)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1809..1828)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2488..3279,3280..3348))
                     /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     3111..3130
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3349..3453)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3521..3539
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3577..3599)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3699..3718
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3787..4242)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3924..3945)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4136..4155
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4368..4390
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4382..4399
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4383..4399
                     /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 atatgcgtgg
      541 cgttcctcat attgttatgg tggacgccta caaacgctat aaatcgggag gtggttcagg
      601 ctacaccatc gtggagcagt acgagagagc cgaggccaga cacagcacct cgaatcctgc
      661 accaggaagt atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
      721 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
      781 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
      841 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc
      901 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
      961 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     1021 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac
     1081 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag
     1141 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac
     1201 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta
     1261 cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc
     1321 agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatg ccgaacaaaa
     1381 actcatctca gaagaggatc tgtagcacca ccaccactaa taaagatctg actgaaaaaa
     1441 aaaaaaaaaa aaagtttaaa cactagtccg ctgagcaata actagcataa ccccttgggg
     1501 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc gggcttcctc
     1561 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1621 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1681 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1741 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1801 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1861 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1921 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     1981 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2041 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2101 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2161 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2221 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2281 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2341 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     2401 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     2461 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     2521 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     2581 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     2641 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     2701 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     2761 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     2821 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     2881 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     2941 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     3001 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     3061 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     3121 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     3181 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     3241 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     3301 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     3361 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     3421 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     3481 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     3541 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3601 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     3661 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     3721 gagagtgcac catatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     3781 atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     3841 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     3901 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     3961 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     4021 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     4081 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     4141 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     4201 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc cattcaggct
     4261 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     4321 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     4381 ttgtaaaacg acggccagtg aattg
//
