LOCUS       sequence-408156-        6151 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of GRFT with N-terminal SpyCatcher fusion and 
            GS linker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6151)
  AUTHORS   Ozkul G, Kehribar ES, Ahan RE, Koksaldi IC, Ozkul A, Dinc B, Aydogan
            S, Seker UOS
  TITLE     A Genetically Engineered Biofilm Material for SARS-CoV-2 Capturing 
            and Isolation.
  JOURNAL   Adv Mater Interfaces. 2022 Sep 13:2201126. doi: 
            10.1002/admi.202201126.
  PUBMED    36248312
REFERENCE   2  (bases 1 to 6151)
  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: "Adv Mater 
            Interfaces. 2022 Sep 13:2201126. doi: 10.1002/admi.202201126."
COMMENT     Sequence Label: pET22b 6His SpyCatcher-GRFT
FEATURES             Location/Qualifiers
     source          1..6151
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        86..163
                     /gene="lacI"
                     /label=lacI promoter
     CDS             164..1246
                     /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(183..202)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2055..2246
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2240..2262)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2348..2487
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2403..2422
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2502..2519)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2673..3261)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2753..2772)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3432..4223,4224..4292))
                     /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     4055..4074
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4293..4397)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4423..4878)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4560..4581)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4772..4791
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4915..4962)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4958..4976
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5029..5046)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5419..5436)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(5452..5790)
                     /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="VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATME
                     LRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     KATKGDAHI"
     CDS             complement(5797..5817)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(5824..5841)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    5885..5909
                     /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(5909..5928)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5910..5928)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6012..6031)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
       61 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat
      121 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
      181 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
      241 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
      301 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
      361 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
      421 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
      481 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
      541 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
      601 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
      661 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
      721 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
      781 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
      841 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
      901 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
      961 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     1021 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     1081 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     1141 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     1201 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     1261 atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca
     1321 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg
     1381 tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg
     1441 aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct
     1501 tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc
     1561 aggccattat cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga
     1621 ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg
     1681 agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg
     1741 tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta
     1801 tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat
     1861 taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg
     1921 ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta
     1981 tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt
     2041 acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca
     2101 gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag
     2161 acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt
     2221 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     2281 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     2341 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     2401 tgcggcatca gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga
     2461 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     2521 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     2581 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     2641 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2701 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2761 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2821 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2881 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2941 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3001 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3061 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     3121 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3181 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3241 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3301 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3361 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     3421 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     3481 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     3541 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     3601 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     3661 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3721 agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt
     3781 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3841 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3901 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     3961 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4021 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4081 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4141 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4201 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4261 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4321 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     4381 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata
     4441 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     4501 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     4561 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     4621 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     4681 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     4741 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     4801 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     4861 cgccgctaca gggcgcgtcc cattcgccaa tccggatata gttcctcctt tcagcaaaaa
     4921 acccctcaag acccgtttag aggccccaag gggttatgct agttattgct cagcggtggc
     4981 agcagccaac tcagcttcct ttcgggcttt gttagcagcc ggatctcagt ggtggtggtg
     5041 gtggtgctcg agttattaat actgttcata ataaatatcc aggctatcca gataatcgcc
     5101 tgcgctaccg ttgatctgga tcaccttcac gttgctcagg gtattggcgc taccaccact
     5161 accaccatac ggaccgaagc ggcgacccat gttggtctcg aagctgatgt tgtcgatata
     5221 gtcgccgctg cggatggtca tgttgctaat gtactcgccg ctgccaaagg taaaggtcgg
     5281 gctcagatta ccaccgctac caccatgatg cacgccatcg atgatgatgg catccaggta
     5341 gctaccactg cgcactgcaa tgctgctcag accgctaaac gggctaccac cgctgccacc
     5401 gaatttgcga tgggtcaggc taccacgcgg aaccagaccg ctgctggatc caatatgagc
     5461 gtcaccttta gttgctttgc catttacagt aacctgacct tgctcattaa ctgtaaaggt
     5521 aatagcagtt gctacctcat aaccgtctgg tgctgcggtt tcgacaaatg tatattttcc
     5581 tggatacagg tagaaatctt tcacttgtcc atctgaaatc catgtactaa tagttttacc
     5641 agatgaatca cgcaactcca tagttgcacc agctaactct ttgccgtcct catcacgttt
     5701 tgagaattta atatgggtag cactatcttc ttcaattgtc atatcaccgg actgaccttg
     5761 ctcacttgat aaacctgata aggtatcaac catggcgccc tgaaaataca ggttttcact
     5821 agtgtggtga tgatgatggt ggctgctacc catggtaccc tccttcttaa agttaatcta
     5881 gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgcgggat
     5941 cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
     6001 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
     6061 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
     6121 gggcgccatc tccttgcatg caccattcct t
//
