LOCUS       sequence-368382-        6063 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of soluble protein tagBFP-sfCherry11-SpyTag 
            with a 6xHIS tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6063)
  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 6063)
  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-6xHIS
FEATURES             Location/Qualifiers
     source          1..6063
                     /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             61..108
                     /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             196..894
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             898..927
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             928..945
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(998..1016)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1012..1059
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1096..1551
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1183..1202)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1393..1414
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1643..2458)
                     /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(1643..2458)
                     /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     2366..2385
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2450..2469
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2580..3168
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3069..3088
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3322..3339
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3354..3493
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3419..3438)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3579..3601
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3595..3786)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4595..5677)
                     /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     5639..5658
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5678..5755)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5961..5980
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactatagga tagtaagtct tgagactaga aagtaaggag gttttttatg
       61 cgtggcgttc ctcatattgt tatggtggac gcctacaaac gctataaatc gggaggtggt
      121 tcaggctaca ccatcgtgga gcagtacgag agagccgagg ccagacacag cacctcgaat
      181 cctgcaccag gaagtatgag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg
      241 gagggcaccg tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac
      301 gagggcaccc agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc
      361 gacatcctgg ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc
      421 atccccgact tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca
      481 tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc
      541 atctacaacg tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag
      601 aaaacactcg gctgggaggc cttcaccgag acgctgtacc ccgctgacgg cggcctggaa
      661 ggcagaaacg acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacatcaag
      721 accacatata gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg
      781 gactacagac tggaaagaat caaggaggcc aacaacgaga cctacgtcga gcagcacgag
      841 gtggcagtgg ccagatactg cgacctccct agcaaactgg ggcacaagct taatgccgaa
      901 caaaaactca tctcagaaga ggatctgcac caccaccacc accactgaga tccggctgct
      961 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1021 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     1081 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     1141 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     1201 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     1261 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     1321 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     1381 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     1441 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     1501 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     1561 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1621 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     1681 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     1741 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     1801 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     1861 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     1921 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     1981 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     2041 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     2101 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     2161 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     2221 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     2281 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     2341 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     2401 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     2461 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     2521 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2581 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2641 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2701 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     2761 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2821 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2881 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2941 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3001 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3061 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3121 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3181 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3241 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3301 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     3361 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     3421 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     3481 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     3541 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     3601 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     3661 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     3721 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     3781 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     3841 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     3901 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     3961 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     4021 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     4081 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     4141 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     4201 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     4261 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     4321 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     4381 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     4441 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     4501 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     4561 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4621 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4681 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     4741 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     4801 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     4861 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     4921 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     4981 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     5041 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     5101 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     5161 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     5221 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     5281 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     5341 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     5401 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     5461 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     5521 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     5581 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     5641 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     5701 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     5761 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     5821 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     5881 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     5941 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     6001 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     6061 aat
//
