LOCUS       sequence-160205-        6067 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial expression vector for split sfCherry screening.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6067)
  AUTHORS   Feng S, Sekine S, Pessino V, Li H, Leonetti MD, Huang B
  TITLE     Improved split fluorescent proteins for endogenous protein labeling.
  JOURNAL   Nat Commun. 2017 Aug 29;8(1):370. doi: 10.1038/s41467-017-00494-8.
  PUBMED    28851864
REFERENCE   2  (bases 1 to 6067)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2017-08-29"; volume: "8(1):370. doi"; pages: " 
            10.1038/s41467-017-00494-8"
COMMENT     Sequence Label: pET_sfCherry(1-10)_32aalinker_sfCherry(11)
FEATURES             Location/Qualifiers
     source          1..6067
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     783..801
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(941..964)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(968..985)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1004..1026
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1041..1065
                     /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)."
     primer_bind     complement(1065..1084)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1066..1084)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1168..1187)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1393..1470
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1471..2553
                     /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     complement(1490..1509)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3362..3553
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3547..3569)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3655..3797
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3710..3729
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3812..3829)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3983..4571)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4063..4082)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4682..4701)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4693..5508
                     /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             4693..5508
                     /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     complement(4766..4785)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5601..6056)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5738..5759)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5950..5969
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagttaggtg ctgtgtctgc
      181 cctcggctct ctcgtactgc tccacgatgg tgtaagaccc cccgccagcg ctccctcccc
      241 cagcagaacc ttcccctccc gaaccagggc ccccgctacc gcctccttct gatccgccac
      301 caccaacgtc gtcctcgttg tggctggtga tgtccagctt gatgtccacg ttgtaggcgc
      361 cgggcagctg cacgggcttc ttggccttgt aggtggtctt cacctcggcg tcgtagtggc
      421 cgccgtcctt cagcttcagt ctctggttga tctcgccctt cagggcgccg tcctcggggt
      481 acattctctc ggtgctggcc tcccagccca tggtcttctt ctgcatcacg gggccgtcgc
      541 tggggaagtt ggtgcccagc agcttcacct tgtagatgaa ctcgccgtcc tgcaggctgc
      601 tgtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcactctc tcccaggtga
      661 agccctcggg gaagctcagc ttcaggtagt cggggatgtc ggcggggtgc ttcacgtagg
      721 ccttgctgcc gtacatgaac tgggggctca ggatgtccca ggcgaagggc agggggccgc
      781 ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggtggccc tcgccctcgc
      841 cctcgatctc gaactcgtgg ccgttcacgc tgccctccat gtgcaccttg aatctcatga
      901 actccttgat gatggccatg ttgtcctcct ccatggatcc gggcccctgg aacagaactt
      961 ccaggctgtg gtgatgatga tgatggctgc tgcccatggt atatctcctt cttaaagtta
     1021 aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt
     1081 attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca
     1141 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
     1201 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
     1261 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
     1321 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
     1381 gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
     1441 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     1501 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     1561 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     1621 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     1681 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     1741 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     1801 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     1861 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     1921 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     1981 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     2041 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     2101 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     2161 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     2221 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     2281 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     2341 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     2401 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     2461 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     2521 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     2581 cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc
     2641 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     2701 gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg
     2761 ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc
     2821 tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc
     2881 cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg
     2941 ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag
     3001 cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg
     3061 tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg
     3121 catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg
     3181 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc
     3241 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct
     3301 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc
     3361 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt
     3421 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc
     3481 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
     3541 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     3601 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     3661 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
     3721 cagattgtac tgagagtgca ccatatatgc ggtgtgaaat accgcacaga tgcgtaagga
     3781 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3841 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3901 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3961 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4021 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4081 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4141 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4201 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4261 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4321 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4381 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4441 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4501 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4561 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4621 actcacgtta agggattttg gtcatgaaca ataaaactgt ctgcttacat aaacagtaat
     4681 acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat
     4741 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     4801 ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     4861 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     4921 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     4981 ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca
     5041 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     5101 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     5161 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     5221 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     5281 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     5341 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     5401 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     5461 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga attaattcat
     5521 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5581 tccccgaaaa gtgccacctg aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa
     5641 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5701 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5761 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     5821 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5881 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     5941 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     6001 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     6061 ttcgcca
//
