LOCUS       sequence-363378-        8726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Display sfGFP protein on E. coli cell surface.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8726)
  AUTHORS   Ahan RE, Kirpat BM, Saltepe B, Seker UOS
  TITLE     A Self-Actuated Cellular Protein Delivery Machine.
  JOURNAL   ACS Synth Biol. 2019 Apr 19;8(4):686-696. doi: 
            10.1021/acssynbio.9b00062. Epub 2019 Mar 12.
  PUBMED    30811932
REFERENCE   2  (bases 1 to 8726)
  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"; doi: 
            "10.1021/acssynbio.9b00062"; journalName: "ACS Synth Biol"; date: 
            "2019-04-19- 19"; volume: "8"; issue: "4"; pages: "686-696"
COMMENT     Sequence Label: pET22b-PelB-6H-Ag43-160N-sfGFP
FEATURES             Location/Qualifiers
     source          1..8726
                     /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"
     CDS             complement(2672..2692)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(2708..3421)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             complement(3437..3454)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     sig_peptide     complement(3461..3526)
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     RBS             3534..3556
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3571..3595
                     /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(3595..3614)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3596..3614)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3698..3717)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3923..4000
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4001..5083
                     /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(4020..4039)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5892..6083
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6077..6099)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6185..6324
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6240..6259
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6339..6356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6510..7098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6590..6609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7269..8060,8061..8129))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7892..7911
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8130..8234)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8260..8715)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8397..8418)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8609..8628
                     /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 gagtcatcag aaggtcacat
      181 tcagtgtggc ctgaccgtta tacccttcag cgctgctgcc gctgacgctg tgggcataac
      241 cggcctgaac gcccagggtg atattttccc ggacacgggc ttccagtccg gcctgcaggt
      301 ccagtgatgt gccattctgt gagggagaga acgtcatccc gctgcctgca gtggaagtcc
      361 ccacacgcat atctccccgg gagctgaagg tgcggataac agaaggctgt acccaccagt
      421 tcaccggtaa ttcactcaca ctgtgttttg cactgtcacg caggggggca cgggatgagg
      481 tgccttcgcc aaaggtcata tcgttgtggc tgcccagacg gaaaccggca cgcacatgtt
      541 gtgcactgcc atgcccgaac ttcacataac cggcgttgtc cttaccgtca tccagggaaa
      601 gtccctgcca ggtatactgc agttgtggct ccagcatcag gttgtcagtg atactgaagg
      661 gcagaccggt ttccagtgag cccagccagc cccagccccg ggcgcggaag tcgttattgt
      721 ccgatgacgc tttcatgctg tggcgggttc cctgtgccac aatgtcagcc cacaggccgg
      781 aggacgtgtg taccagattc aggtatccgc ccaggctgcc ggcatcatcc cggaccgtgc
      841 cggcacggga gccgtcatca tccttaacat caacggaaga atggccagca gcaccatata
      901 cccccgcggt cacagacata ccggcaacct ctgttctcat caggtcaccc tccagacgga
      961 cgaatccata gctgccgctg ctttccggcg tggccccacg ggcaataccg ccattgttat
     1021 cgtgaccgag atgaccgccc tgaatgctga gacggacgct gttgttttca ccatttacac
     1081 cggtctgatg gctgcgggag cctgccacaa tccggtcata gtccattgcc tgtgtcagca
     1141 tggaggcata cagggggact tctgcacgat aagcattttc actgcgcaga taccagctct
     1201 catcactgtc ccggttgagg gagtagttaa aggcaccggc ctgcagcctg ttcccctgga
     1261 caaaggcccc ttcctccgtg gtggcaccgt taatggcttc caccacctga atacccttac
     1321 cgctggtcgc cagccccgac gcactgttgc cggcgttcac caggttcagg atggtttttc
     1381 cggttgccct gccgccgtca atgaccagtc tgtcagcatt gttctgtgcc atatccgggc
     1441 gtacacgcag gctgatggtg ccattctgtc cgttcaggtt tttcactttc agggttgccg
     1501 gtacgaactt ccctgtgcgg gtggaggtga aatgaatctg tccggcatgg ctgaggtcat
     1561 ccaccaccga ctgcaccgtg gcgttatcgg ggatattcca ggtggcaccg gaggcgagag
     1621 tgacattcgt ggggtcaatg gcaccgttca gcacagtgct gccggtcagc ttcagggctg
     1681 taccgcgctg agcaatgaca tccgtggtga cggtactgtc gttcagcgtc agcgtgcctt
     1741 cattcaggtt gacggctttc tgggtgagtg tggtgttgct gacagtgagt gtgccacttc
     1801 ctgatttttc cacgctgccg ttaccggtga gagagcctcc ctgcaggagt gcatcgcctt
     1861 cacggatggt cagggtatcg ttgttcaccg tcttcccgga aagggtaagt atggcgccgt
     1921 tattcagcgt ggtggtgccc gccagtgtgc cgcccctggc ggtgaacagt ccgccattta
     1981 ccgtggtatc cgtggccgta tcaccggcgt tcagcgtgaa tgaactgcct ttttccagca
     2041 tcagggcatc cgcctgaccg cctccgatac tgaatgcctt tccgtcgctc cgggtaccac
     2101 tgacagcggc accggaatcg gccagcagta caccgccatt tcccatggat acggtggtgg
     2161 cggtgccacc gttgcggaca tccagcgttc cgccatcatc cacgcgggta ttagtggctg
     2221 tgtgtccggt cagcacatcc aggcgtccgc cattttccag tacgacatta tcagctttac
     2281 cctccacaac agagaatgct cccaggcggt ttatgccggt aacggttgca gccgtactgg
     2341 taaccagtgc gccgccctgc ttcagggtga cattcgtggc tgtaccaccg gcgtccacct
     2401 gcagtctgcc cttctgatta acggtggtat tcccggccac acccccgttt ttgacaatct
     2461 gccagccgtc actgttcaca acagtgtcag acgctgtacc gccgttgtgc acatactggt
     2521 atcccccatt cagcgtggta tccagtgcgt gaccatgtac agtctggtcg ccgccggcat
     2581 aaaccacagt ggtatttgcc gttccttcag ctctcacaat ctggcgaccg tttttattga
     2641 tggttgtgcg cttaaggcta ccaccgccac cgccctgaaa gtacaggttt tcgctaccac
     2701 cgccaccttt gtacagttca tccataccat gcgtgatgcc cgctgcggtt acgaactcca
     2761 gcagaaccat atgatcgcgt ttctcgttcg gatctttaga cagaacgctt tgcgtgctca
     2821 gatagtgatt gtctggcagc agaacaggac catcaccgat tggagtgttt tgctggtagt
     2881 gatcagccag ctgcacgctg ccatcctcca cgttgtggcg aattttaaaa ttcgctttaa
     2941 tgccattttt ttgtttatcg gcggtgatgt aaacattgtg gctgttaaaa ttgtattcca
     3001 gcttatggcc caggatattg ccgtcttctt taaagtcaat gcctttcagc tcaatgcggt
     3061 ttaccagggt atcgccttca aatttcactt ccgcacgcgt tttgtacgtg ccgtcatcct
     3121 taaaggaaat cgtgcgttcc tgcacatagc cttccggcat ggcggacttg aagaagtcat
     3181 gctgcttcat atggtccgga taacgagcaa agcactgaac accataagtc agcgtcgtta
     3241 ccagagtcgg ccaaggtacc ggcagtttac cagtagtaca gatgaacttc agcgtcagtt
     3301 taccattagt tgcgtcacct tcaccctcgc cacgcacgga aaacttatga ccgttgacat
     3361 caccatccag ttccaccaga atagggacga caccagtgaa cagctcttcg cctttacgca
     3421 tggatccacc gccaccgtgg tggtggtggt ggtggcccat ggccatcgcc ggctgggcag
     3481 cgaggagcag cagaccagca gcagcggtcg gcagcaggta tttcatatgt atatctcctt
     3541 cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat
     3601 agtgagtcgt attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg
     3661 gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat
     3721 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
     3781 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
     3841 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
     3901 aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc
     3961 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     4021 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     4081 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     4141 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     4201 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     4261 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     4321 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     4381 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     4441 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     4501 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     4561 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     4621 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     4681 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     4741 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     4801 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     4861 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     4921 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     4981 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     5041 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     5101 taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc
     5161 cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct
     5221 tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg
     5281 accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc
     5341 acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg
     5401 ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga
     5461 cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc
     5521 gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct
     5581 tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt
     5641 tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa
     5701 cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca
     5761 gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg
     5821 tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca
     5881 cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa
     5941 gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca
     6001 tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg
     6061 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     6121 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     6181 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     6241 ggcatcagag cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc
     6301 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     6361 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     6421 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6481 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6541 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6601 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6661 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6721 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6781 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6841 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6901 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     6961 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     7021 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     7081 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     7141 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     7201 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     7261 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     7321 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     7381 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     7441 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     7501 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7561 ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg
     7621 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7681 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7741 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7801 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7861 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7921 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7981 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     8041 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     8101 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     8161 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     8221 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     8281 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     8341 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     8401 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     8461 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     8521 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     8581 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     8641 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     8701 cgctacaggg cgcgtcccat tcgcca
//
