LOCUS       sequence-226764-        6548 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector for split sfCherry3 screening. The 
            "sfCherry2(1-10)" and "sfCherry2(11)-SpyCatcher002" are expressed 
            from two different T7 promoters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6548)
  AUTHORS   Feng S, Varshney A, Coto Villa D, Modavi C, Kohler J, Farah F, Zhou 
            S, Ali N, Muller JD, Van Hoven MK, Huang B
  TITLE     Bright split red fluorescent proteins for the visualization of 
            endogenous proteins and synapses.
  JOURNAL   Commun Biol. 2019 Sep 17;2:344. doi: 10.1038/s42003-019-0589-x. 
            eCollection 2019.
  PUBMED    31552297
REFERENCE   2  (bases 1 to 6548)
  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: "Commun 
            Biol."; date: "2019-09-17"; volume: "2:344. doi"; pages: " 
            10.1038/s42003-019-0589-x. eCollection 2019"
COMMENT     Sequence Label: pETDuet_sfCherry2(1-10)_sfCherry2(11)-SpyCatcher
FEATURES             Location/Qualifiers
     source          1..6548
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(204..222)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             711..728
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        842..860
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    861..885
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     CDS             1022..1039
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1061..1081
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1088..1426
                     /codon_start=1
                     /product="truncated version of the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyCatcher
                     /note="forms a covalent isopeptide bond with the SpyTag 
                     peptide (Zakeri et al., 2012)"
                     /translation="VDTLSGLSCEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATME
                     LRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     KATKGDAHI"
     CDS             1497..1541
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1579..1597)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1593..1640
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1677..2132
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1764..1783)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1974..1995
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2247..3038,3039..3107))
                     /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     2870..2889
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3108..3200)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3281..3869
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3770..3789
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4023..4040
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4055..4194
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4120..4139)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4280..4302
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4296..4487)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5059..6141)
                     /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     6103..6122
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6142..6219)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6425..6444
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6532..6548,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atggaggagg acaacatggc catcatcaag gagttcatga gattcaaggt
      121 gcacatggag ggcagcgtga acggccacga gttcgagatc gagggcgagg gcgagggcca
      181 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt
      241 cgcctgggac atcctgagcc cccagttcat gtacggcagc aaggcctacg tgaagcaccc
      301 cgccgacatc cccgactacc tgaagctgag cttccccgag ggcttcacct gggagagagt
      361 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gacagcagcc tgcaggacgg
      421 ccagttcatc tacaaggtga agctgctggg catcaacttc cccagcgacg gccccgtgat
      481 gcagaagaag accatgggct gggaggccag caccgagaga atgtaccccg aggacggcgc
      541 cctgaagggc gagatcaacc agagactgaa gctgaaggac ggcggccact acgacgccga
      601 ggtgaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtgga
      661 catcaagctg gacatcacca gccacaacga ggactgacat gggcagcagc catcaccatc
      721 atcaccacag ccaggatccg aattcgagct cggcgcgcct gcaggtcgac aagcttgcgg
      781 ccgcataatg cttaagtcga acagaaagta atcgtattgt acacggccgc ataatcgaaa
      841 ttaatacgac tcactatagg ggaattgtga gcggataaca attccccatc ttagtatatt
      901 agttaagtat aagaaggaga tatacataat gtacaccatc gtggagcagt acgagagagc
      961 cgaggccaga cacagcaccg tccggctcag atctggaggc tccggaggca tgtcgtacta
     1021 ccatcaccat caccatcacg attacgacat cccaacgacc gaaaacctgt attttcaggg
     1081 cgccatggtt gataccttat caggtttatc atgtgagcaa ggtcagtccg gtgatatgac
     1141 aattgaagaa gatagtgcta cccatattaa attctcaaaa cgtgatgagg acggcaaaga
     1201 gttagctggt gcaactatgg agttgcgtga ttcatctggt aaaactatta gtacatggat
     1261 ttcagatgga caagtgaaag atttctacct gtatccagga aaatatacat ttgtcgaaac
     1321 cgcagcacca gacggttatg aggtagcaac tgctattacc tttacagtta atgagcaagg
     1381 tcaggttact gtaaatggca aagcaactaa aggtgacgct catatttaat atggcagatc
     1441 tcaattggat atcggccggc cacgcgatcg ctgacgtcgg taccctcgag tctggtaaag
     1501 aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat
     1561 taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1621 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     1681 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1741 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1801 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1861 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1921 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1981 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2041 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2101 gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat gagattatca
     2161 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2221 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2281 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2341 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     2401 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     2461 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     2521 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     2581 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     2641 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     2701 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     2761 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     2821 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     2881 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     2941 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3001 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3061 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3121 caatcatgat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     3181 tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3241 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3301 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3361 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3421 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3481 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3541 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3601 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3661 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3721 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3781 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3841 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3901 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3961 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4021 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     4081 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     4141 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     4201 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4261 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4321 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     4381 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     4441 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     4501 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     4561 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     4621 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     4681 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     4741 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     4801 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     4861 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4921 ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact
     4981 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     5041 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5101 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     5161 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     5221 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     5281 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     5341 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     5401 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     5461 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     5521 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     5581 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     5641 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5701 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5761 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5821 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5881 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5941 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     6001 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     6061 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     6121 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     6181 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     6241 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     6301 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     6361 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     6421 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     6481 ccacgatgcg tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac
     6541 tcactata
//
