LOCUS       sequence-338549-        6072 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses in bacterial cytoplasm superfolder GFP with DogTag and 
            flanking linkers between Val22 and Asn23.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6072)
  AUTHORS   Keeble AH, Yadav VK, Ferla MP, Bauer CC, Chuntharpursat-Bon E, Huang
            J, Bon RS, Howarth M
  TITLE     DogCatcher allows loop-friendly protein-protein ligation.
  JOURNAL   Cell Chem Biol. 2021 Jul 28. pii: S2451-9456(21)00315-9. doi: 
            10.1016/j.chembiol.2021.07.005.
  PUBMED    34324879
REFERENCE   2  (bases 1 to 6072)
  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: "Cell Chem 
            Biol. 2021 Jul 28. pii: S2451-9456(21)00315-9. doi: 
            10.1016/j.chembiol.2021.07.005."
COMMENT     Sequence Label: pET28-sfGFP-DogTag Loop A
FEATURES             Location/Qualifiers
     source          1..6072
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(778..1860)
                     /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     1822..1841
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1861..1938)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2144..2163
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2247..2266
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2247..2265
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2266..2290
                     /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             2305..2327
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2416..2484
                     /codon_start=1
                     /product="peptide derived from the RrgA adhesin of 
                     Streptococcus pneumoniae"
                     /label=DogTag
                     /note="forms a covalent bond with the DogCatcher protein 
                     when inserted into an intra-domain protein loop (Keeble et 
                     al., 2022)"
                     /translation="DIPATYEFTDGKHYITNEPIPPK"
     CDS             3166..3183
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3253..3271)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3267..3314
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3351..3806
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3438..3457)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3648..3669
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3898..4713)
                     /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(3898..4713)
                     /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     4621..4640
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4705..4724
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4835..5423
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5324..5343
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5577..5594
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5609..5748
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5674..5693)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5834..5856
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5850..6041)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
       61 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
      121 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
      181 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
      241 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
      301 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
      361 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
      421 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
      481 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
      541 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
      601 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
      661 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
      721 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
      781 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
      841 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
      901 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
      961 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     1021 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     1081 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     1141 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     1201 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     1261 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     1321 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     1381 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     1441 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     1501 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     1561 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     1621 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     1681 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     1741 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     1801 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     1861 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     1921 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     1981 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     2041 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     2101 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     2161 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     2221 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     2281 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgcgt
     2341 aaaggcgaag agctgttcac tggtgtcgtc cctattctgg tggaactgga tggtgatgtc
     2401 ggaggaggtg gctctgatat tccggctaca tacgaattca ccgatggtaa acattatatc
     2461 accaatgaac cgataccgcc gaaaggcggg ggtggctcga acggtcataa gttttccgtg
     2521 cgtggcgagg gtgaaggtga cgcaactaat ggtaaactga cgctgaagtt catctgtact
     2581 actggtaaac tgccggtacc ttggccgact ctggtaacga cgctgactta tggtgttcag
     2641 tgctttgctc gttatccgga ccatatgaag cagcatgact tcttcaagtc cgccatgccg
     2701 gaaggctatg tgcaggaacg cacgatttcc tttaaggatg acggcacgta caaaacgcgt
     2761 gcggaagtga aatttgaagg cgataccctg gtaaaccgca ttgagctgaa aggcattgac
     2821 tttaaagaag acggcaatat cctgggccat aagctggaat acaattttaa cagccacaat
     2881 gtttacatca ccgccgataa acaaaaaaat ggcattaaag cgaattttaa aattcgccac
     2941 aacgtggagg atggcagcgt gcagctggct gatcactacc agcaaaacac tccaatcggt
     3001 gatggtcctg ttctgctgcc agacaatcac tatctgagca cgcaaagcgt tctgtctaaa
     3061 gatccgaacg agaaacgcga tcatatggtt ctgctggagt tcgtaaccgc agcgggcatc
     3121 acgcatggta tggatgaact gtacaaaggc tccggctccg gctcccacca tcaccatcac
     3181 cattgataag ctagcaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     3241 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     3301 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     3361 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3421 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3481 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3541 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3601 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3661 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3721 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     3781 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     3841 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     3901 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     3961 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     4021 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     4081 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     4141 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     4201 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     4261 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     4321 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     4381 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     4441 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     4501 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     4561 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     4621 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     4681 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     4741 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4801 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4861 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4921 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4981 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5041 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5101 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5161 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5221 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5281 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5341 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5401 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5461 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5521 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5581 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     5641 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5701 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     5761 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5821 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5881 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     5941 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     6001 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     6061 ggatttctgt tc
//
