LOCUS       sequence-337383-        5872 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses anticalin targeting CTLA-4 with Fg-beta peptide at the N 
            terminus and 6x-His tag and ybbr tag at the C terminus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5872)
  AUTHORS   Liu Z, Moreira RA, Dujmovic A, Liu H, Yang B, Poma AB, Nash MA
  TITLE     Mapping Mechanostable Pulling Geometries of a Therapeutic 
            Anticalin/CTLA-4 Protein Complex.
  JOURNAL   Nano Lett. 2022 Jan 12;22(1):179-187. doi: 
            10.1021/acs.nanolett.1c03584. Epub 2021 Dec 17.
  PUBMED    34918516
REFERENCE   2  (bases 1 to 5872)
  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/acs.nanolett.1c03584"; journalName: "Nano Lett"; date: 
            "2022-01-12- 12"; volume: "22"; issue: "1"; pages: "179-187"
COMMENT     Sequence Label: pET28a-Fg-beta-anticalin-His-ybbr
FEATURES             Location/Qualifiers
     source          1..5872
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      29..484
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(116..135)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     326..347
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(576..1391)
                     /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(576..1391)
                     /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     1299..1318
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1383..1402
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1513..2101
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2002..2021
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2255..2272
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2287..2426
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2352..2371)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2512..2534
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2528..2719)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3528..4610)
                     /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     4572..4591
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4611..4688)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4894..4913
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4997..5016
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4997..5015
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5016..5040
                     /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             5055..5077
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5685..5702
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5803..5821)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5817..5864
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
       61 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
      121 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
      181 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
      241 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
      301 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
      361 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
      421 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
      481 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      541 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
      601 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
      661 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
      721 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
      781 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
      841 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
      901 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
      961 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     1021 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     1081 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     1141 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     1201 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     1261 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     1321 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     1381 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     1441 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     1501 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     1561 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     1621 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     1681 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     1741 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     1801 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     1861 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     1921 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     1981 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2041 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     2101 aaacgccagc aacgcggcct ttttacggtt cccggccttt tgctggcctt ttgctcacat
     2161 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     2221 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     2281 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     2341 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     2401 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     2461 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     2521 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     2581 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     2641 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     2701 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     2761 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     2821 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     2881 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     2941 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     3001 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     3061 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     3121 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     3181 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     3241 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     3301 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     3361 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     3421 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     3481 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     3541 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3601 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     3661 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     3721 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     3781 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     3841 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     3901 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     3961 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     4021 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     4081 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     4141 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     4201 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     4261 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     4321 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     4381 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     4441 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     4501 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     4561 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     4621 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     4681 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     4741 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     4801 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     4861 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     4921 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     4981 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     5041 cctgtagaaa taattttgtt taactttaag aaggagatat acatatgttt tttagcgcac
     5101 gtggtcatcg tccgctggat ggtagcggta gccaggatag caccagcgat ctgattccgg
     5161 caccgcctct gagcaaagtt ccgctgcagc agaattttca ggataatcag tttcacggca
     5221 aatggtatgt tgttggtctg gcaggtaatc gtattctgcg tgatgatcag catccgatga
     5281 atatgtatgc caccatctat gaactgaaag aggacaaaag ctataacgtg accagcgtta
     5341 ttagcagcca caaaaaatgc gaatatacca ttgcaacctt tgttccgggt agccagcctg
     5401 gtgaatttac cctgggtaac attaaaagct atggcgataa aaccagctat ctggttcgtg
     5461 ttgttagcac cgattataac cagtatgccg tggttttttt caaactggcc gaagataatg
     5521 ccgaattttt tgccattacc atttacggtc gtaccaaaga actggcaagc gagctgaaag
     5581 aaaactttat tcgttttagc aaaagcctgg gtctgccgga aaatcatatt gtgtttccgg
     5641 ttccgattga tcagtgtatt gatggtggta gcggtagcgg ttcacatcat catcaccatc
     5701 atggtagcga ctctctggaa ttcatcgctt ctaaactggc ttaactcgag taagatccgg
     5761 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     5821 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa gg
//
