LOCUS       sequence-368392-        4561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free expression of transmembrane protein 
            PDGFR-tagBFP-sfCherry11-SpyTag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4561)
  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 4561)
  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: InterTag-BFP
FEATURES             Location/Qualifiers
     source          1..4561
                     /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"
     CDS             1406..1552
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     primer_bind     complement(1624..1642)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1638..1685
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1714..1731)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1885..2473)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1965..1984)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2644..3435,3436..3504))
                     /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     3267..3286
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3505..3609)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3677..3695
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3733..3755)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3855..3874
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3943..4398)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4080..4101)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4292..4311
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4524..4546
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4538..4555
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4539..4555
                     /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 tgaatgctgt gggccaggac acgcaggagg tcatcgtggt
     1441 gccacactcc ttgcccttta aggtggtggt gatctcagcc atcctggccc tggtggtgct
     1501 caccatcatc tcccttatca tcctcatcat gctttggcag aagaagccac gttagcacca
     1561 ccaccactaa taaagatctg actgaaaaaa aaaaaaaaaa aaaaaaaaag tttaaacact
     1621 agtccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     1681 ttttgctgaa aggaggaact atatccgggc ttcctcgctc actgactcgc tgcgctcggt
     1741 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     1801 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     1861 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     1921 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     1981 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2041 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     2101 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     2161 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     2221 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     2281 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     2341 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     2401 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     2461 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     2521 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     2581 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     2641 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     2701 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     2761 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     2821 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     2881 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     2941 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3001 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3061 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3121 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3181 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     3241 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     3301 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3361 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3421 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3481 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     3541 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     3601 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     3661 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga
     3721 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     3781 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     3841 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tatgcggtgt
     3901 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta agcgttaata
     3961 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     4021 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     4081 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     4141 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     4201 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     4261 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     4321 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     4381 cgccgctaca gggcgcgtcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     4441 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     4501 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
     4561 g
//
