LOCUS       sequence-337387-        5788 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses anticalin targeting CTLA-4 with the codon encoding S87 
            changed to amber codon (TAG)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5788)
  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 5788)
  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-anticalin (S87amb)-His
FEATURES             Location/Qualifiers
     source          1..5788
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      22..477
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(109..128)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     319..340
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(569..1384)
                     /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(569..1384)
                     /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     1292..1311
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1376..1395
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1506..2094
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1995..2014
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2248..2265
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2280..2419
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2345..2364)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2505..2527
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2521..2712)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3521..4603)
                     /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     4565..4584
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4604..4681)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4887..4906
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4990..5009
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4990..5008
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5009..5033
                     /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             5048..5070
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5633..5650
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5712..5730)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5726..5773
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
       61 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
      121 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
      181 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
      241 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
      301 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
      361 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
      421 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
      481 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
      541 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
      601 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
      661 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
      721 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
      781 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
      841 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
      901 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
      961 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     1021 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     1081 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     1141 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     1201 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     1261 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     1321 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     1381 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     1441 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1501 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1561 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1621 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     1681 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1741 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     1801 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     1861 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     1921 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     1981 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2041 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2101 agcaacgcgg cctttttacg gttcccggcc ttttgctggc cttttgctca catgttcttt
     2161 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2221 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2281 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     2341 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     2401 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     2461 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     2521 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     2581 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     2641 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     2701 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     2761 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     2821 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     2881 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     2941 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     3001 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     3061 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     3121 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     3181 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     3241 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     3301 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     3361 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     3421 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     3481 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3541 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3601 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     3661 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     3721 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     3781 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     3841 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     3901 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     3961 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     4021 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     4081 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     4141 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     4201 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     4261 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     4321 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     4381 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     4441 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     4501 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     4561 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     4621 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     4681 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     4741 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     4801 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     4861 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     4921 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     4981 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctgtag
     5041 aaataatttt gtttaacttt aagaaggaga tatacatatg caggatagca ccagcgatct
     5101 gattccggca ccgcctctga gcaaagttcc gctgcagcag aattttcagg ataatcagtt
     5161 tcacggcaaa tggtatgttg ttggtctggc aggtaatcgt attctgcgtg atgatcagca
     5221 tccgatgaat atgtatgcca ccatctatga actgaaagag gacaaaagct ataacgtgac
     5281 cagcgttatt agcagccaca aaaaatgcga atataccatt gcaacctttg ttccgggtta
     5341 gcagcctggt gaatttaccc tgggtaacat taaaagctat ggcgataaaa ccagctatct
     5401 ggttcgtgtt gttagcaccg attataacca gtatgccgtg gtttttttca aactggccga
     5461 agataatgcc gaattttttg ccattaccat ttacggtcgt accaaagaac tggcaagcga
     5521 gctgaaagaa aactttattc gttttagcaa aagcctgggt ctgccggaaa atcatattgt
     5581 gtttccggtt ccgattgatc agtgtattga tggtggtagc ggtagcggtt cacatcatca
     5641 tcaccatcat taactcgagt aagatccggc tgctaacaaa gcccgaaagg aagctgagtt
     5701 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     5761 gaggggtttt ttgctgaaag gaggaact
//
