LOCUS       sequence-341563-        6846 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of eGFP-CBP protein for chitin visualization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6846)
  AUTHORS   Ricardo-Gonzalez RR, Kotas ME, Tenvooren I, Marquez DM, Fassett MS, 
            Lee J, Daniel SG, Bittinger K, Diaz RE, Fraser JS, Ansel KM, Spitzer
            MH, Liang H, Locksley RM
  TITLE     Type 2 innate immunity regulates hair follicle homeostasis to 
            control Demodex pathosymbionts
  JOURNAL   bioRxiv 2021.03.29.437438
REFERENCE   2  (bases 1 to 6846)
  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: "bioRxiv 
            2021.03.29.437438"
COMMENT     Sequence Label: eGFP-CBP
FEATURES             Location/Qualifiers
     source          1..6846
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..6,7..717)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             754..1347
                     /codon_start=1
                     /gene="Mycobacterium xenopi gyrA"
                     /product="modified GyrA intein (Southworth et al., 1999)"
                     /label=Mxe GyrA intein
                     /note="N-terminal cleavage of this intein can be induced by
                     thiols."
                     /translation="CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPV
                     LADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAV
                     IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY
                     AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA"
     CDS             1378..1533
                     /codon_start=1
                     /gene="Bacillus circulans chiA"
                     /product="chitin binding domain from chitinase A1 (Watanabe
                     et al., 1994)"
                     /label=CBD
                     /translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
                     PALWQLQ"
     CDS             1534..1551
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1561..1578
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1631..1649)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1645..1692
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1729..2184
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1816..1835)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2026..2047
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2276..3091)
                     /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(2276..3091)
                     /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     2999..3018
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3083..3102
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3213..3801
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3702..3721
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3955..3972
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3987..4126
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4052..4071)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4212..4234
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4228..4419)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5228..6310)
                     /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     6272..6291
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6311..6388)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6594..6613
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6697..6716
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6697..6715
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6716..6740
                     /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             6755..6777
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6796..6813
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6823..6840
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 atggttagca aaggcgaaga actgtttacg ggcgtggtgc cgatcctggt ggaactggac
       61 ggtgatgtta atggccataa attttcagtc tcgggtgaag gcgaaggtga tgccacctat
      121 ggtaaactga cgctgaaatt catttgcacc acgggtaaac tgccggtccc gtggccgacc
      181 ctggtgacca cgctgacgta tggtgtgcaa tgtttttctc gttacccgga tcacatgaaa
      241 cagcacgact ttttcaaaag tgccatgccg gaaggttatg ttcaggaacg caccattttc
      301 tttaaagatg acggcaacta caaaacccgt gcagaagtca aatttgaagg tgatacgctg
      361 gtgaaccgca ttgaactgaa aggcattgat ttcaaagaag acggtaatat cctgggccat
      421 aaactggaat ataactacaa tagccacaac gtgtatatta tggccgataa acagaaaaac
      481 ggtatcaaag ttaacttcaa aatccgtcat aatatcgaag atggctctgt ccagctggca
      541 gaccactatc agcaaaacac cccgattggc gatggtccgg tgctgctgcc ggacaatcat
      601 tacctgagca cgcagtctgc gctgagtaaa gatccgaacg aaaaacgcga ccacatggtt
      661 ctgctggagt tcgtcaccgc ggccggtatt acgctgggca tggatgaact gtataaaggt
      721 ggacctggag gatccataat tcacctcgag tactgcatca cgggagatgc actagttgcc
      781 ctacccgagg gcgagtcggt acgcatcgcc gacatcgtgc cgggtgcgcg gcccaacagt
      841 gacaacgcca tcgacctgaa agtccttgac cggcatggca atcccgtgct cgccgaccgg
      901 ctgttccact ccggcgagca tccggtgtac acggtgcgta cggtcgaagg tctgcgtgtg
      961 acgggcaccg cgaaccaccc gttgttgtgt ttggtcgacg tcgccggggt gccgaccctg
     1021 ctgtggaagc tgatcgacga aatcaagccg ggcgattacg cggtgattca acgcagcgca
     1081 ttcagcgtcg actgtgcagg ttttgcccgc gggaaacccg aatttgcgcc cacaacctac
     1141 acagtcggcg tccctggact ggtgcgtttc ttggaagcac accaccgaga cccggacgcc
     1201 caagctatcg ccgacgagct gaccgacggg cggttctact acgcgaaagt cgccagtgtc
     1261 accgacgccg gcgtgcagcc ggtgtatagc cttcgtgtcg acacggcaga ccacgcgttt
     1321 atcacgaacg ggttcgtcag ccacgctact ggcctcaccg gtctgaactc aggcctcacg
     1381 acaaatcctg gtgtatccgc ttggcaggtc aacacagctt atactgcggg acaattggtc
     1441 acatataacg gcaagacgta taaatgtttg cagccccaca cctccttggc aggatgggaa
     1501 ccatccaacg ttcctgcctt gtggcagctt caacatcacc atcaccatca ctaactcgag
     1561 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     1621 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1681 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     1741 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1801 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1861 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1921 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1981 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2041 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2101 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2161 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2221 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     2281 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     2341 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     2401 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     2461 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     2521 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     2581 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     2641 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     2701 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     2761 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     2821 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     2881 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     2941 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     3001 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     3061 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     3121 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3181 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3241 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3301 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     3361 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3421 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3481 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3541 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3601 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3661 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3721 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3781 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3841 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3901 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3961 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     4021 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4081 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     4141 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4201 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4261 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     4321 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     4381 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     4441 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     4501 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     4561 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     4621 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     4681 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     4741 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     4801 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     4861 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     4921 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     4981 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     5041 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     5101 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     5161 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     5221 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5281 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     5341 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     5401 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     5461 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     5521 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     5581 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     5641 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     5701 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     5761 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     5821 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     5881 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     5941 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     6001 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     6061 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     6121 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     6181 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     6241 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     6301 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     6361 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     6421 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     6481 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     6541 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     6601 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     6661 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     6721 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     6781 accatgggca gcagccatca tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc
     6841 catatg
//
