LOCUS       sequence-338612-        5109 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express DogCatcher-superfolder GFP protein in bacterial cytoplasm.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5109)
  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 5109)
  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: pJ404-DogCatcher-sfGFP
FEATURES             Location/Qualifiers
     source          1..5109
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     858..875
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     859..875
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1331..1347
                     /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)."
     promoter        join(1372..1386,1387..1392,1393..1409,1410..1416)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1392..1408
                     /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)."
     protein_bind    1422..1441
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     CDS             1510..1527
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1549..1569
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1576..1887
                     /codon_start=1
                     /product="engineered fragment of the RrgA adhesin of 
                     Streptococcus pneumoniae"
                     /label=DogCatcher
                     /note="forms a covalent bond with an intra-domain protein 
                     loop containing the DogTag peptide (Keeble et al., 2022)"
                     /translation="KLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTY
                     QDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQ
                     "
     CDS             1903..2613
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="RKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2821..2840)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2846..2865)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2907..2998
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2999..3067,3068..3859)
                     /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     complement(3217..3236)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             3942..5024
                     /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(3961..3980)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag cccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag gcgagagtag
      781 ggaactgcca ggcatcaaac taagcagaag gcccctgacg gatggccttt ttgcgtttct
      841 acaaactctt tctgtgttgt aaaacgacgg ccagtcttaa gctcgggccc cctgggcggt
      901 tctgataacg agtaatcgtt aatccgcaaa taacgtaaaa acccgcttcg gcgggttttt
      961 ttatgggggg agtttaggga aagagcattt gtcagaatat ttaagggcgc ctgtcacttt
     1021 gcttgatata tgagaattat ttaaccttat aaatgagaaa aaagcaacgc actttaaata
     1081 agatacgttg ctttttcgat tgatgaacac ctataattaa actattcatc tattatttat
     1141 gattttttgt atatacaata tttctagttt gttaaagaga attaagaaaa taaatctcga
     1201 aaataataaa gggaaaatca gtttttgata tcaaaattat acatgtcaac gataatacaa
     1261 aatataatac aaactataag atgttatcag tatttattat catttagaat aaattttgtg
     1321 tcgcccttaa ttgtgagcgg ataacaatta cgagcttcat gcacagtgaa atcatgaaaa
     1381 atttatttgc tttgtgagcg gataacaatt ataatatgtg gaattgtgag cgctcacaat
     1441 tccacaacgg tttccctgta gaaataattt tgtttaactt taataaggag atataccatg
     1501 tcgtactacc atcaccatca ccatcacgat tacgacatcc caacgaccga aaacctgtat
     1561 tttcagggcg ccatgaaact gggcgagatt gaatttatta aagtggacaa aaccgataaa
     1621 aagccgctgc gtggtgccgt gtttagcctg cagaaacagc atcccgacta tcccgatatc
     1681 tatggcgcga ttgatcagaa tgggacctat caagatgtgc gtaccggcga agatggtaaa
     1741 ctgaccttta cgaatctgag cgatggcaaa tatcgcctga ttgaaaatag cgaacccccg
     1801 ggctataaac cggtgcagaa taagccgatt gtgagctttc gtattgtgga tggcgaagtg
     1861 cgtgatgtga ccagtattgt gccgcaggga tccagtggta gccgtaaagg cgaagagctg
     1921 ttcactggtg tcgtccctat tctggtggaa ctggatggtg atgtcaacgg tcataagttt
     1981 tccgtgcgtg gcgagggtga aggtgacgca actaatggta aactgacgct gaagttcatc
     2041 tgtactactg gtaaactgcc ggtaccttgg ccgactctgg taacgacgct gacttatggt
     2101 gttcagtgct ttgctcgtta tccggaccat atgaagcagc atgacttctt caagtccgcc
     2161 atgccggaag gctatgtgca ggaacgcacg atttccttta aggatgacgg cacgtacaaa
     2221 acgcgtgcgg aagtgaaatt tgaaggcgat accctggtaa accgcattga gctgaaaggc
     2281 attgacttta aagaagacgg caatatcctg ggccataagc tggaatacaa ttttaacagc
     2341 cacaatgttt acatcaccgc cgataaacaa aaaaatggca ttaaagcgaa ttttaaaatt
     2401 cgccacaacg tggaggatgg cagcgtgcag ctggctgatc actaccagca aaacactcca
     2461 atcggtgatg gtcctgttct gctgccagac aatcactatc tgagcacgca aagcgttctg
     2521 tctaaagatc cgaacgagaa acgcgatcat atggttctgc tggagttcgt aaccgcagcg
     2581 ggcatcacgc atggtatgga tgaactgtac aaatgataac tcgagcccca agggcgacac
     2641 cccataatta gcccgggcga aaggcccagt ctttcgactg agcctttcgt tttatttgat
     2701 gcctggcagt tccctactct cgcatgggga gtccccacac taccatcggc gctacggcgt
     2761 ttcacttctg agttcggcat ggggtcaggt gggaccaccg cgctactgcc gccaggcaaa
     2821 caaggggtgt tatgagccat attcaggtat aaatgggctc gcgataatgt tcagaattgg
     2881 ttaattggtt gtaacactga cccctatttg tttatttttc taaatacatt caaatatgta
     2941 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagaatat
     3001 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3061 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     3121 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     3181 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     3241 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     3301 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     3361 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     3421 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     3481 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3541 tgtagcgatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3601 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3661 ggcccttccg gctggctggt ttattgctga taaatccgga gccggtgagc gtggttctcg
     3721 cggtatcatc gcagcgctgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3781 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3841 actgattaag cattggtaag cggcgcgcca tcgaatggcg caaaaccttt cgcggtatgg
     3901 catgatagcg cccggaagag agtcaattca gggtggtgaa tatgaaacca gtaacgttat
     3961 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     4021 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     4081 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     4141 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     4201 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     4261 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     4321 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     4381 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaggacggta
     4441 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     4501 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     4561 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     4621 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     4681 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     4741 atatctcggt agtgggatac gacgataccg aagatagctc atgttatatc ccgccgttaa
     4801 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     4861 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc agtctcactg gtgaaaagaa
     4921 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     4981 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgactcatg accaaaatcc
     5041 cttaacgtga gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5101 aggatcttc
//
