LOCUS       sequence-337384-        5788 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses anticalin targeting CTLA-4 with the codon encoding N21 
            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 (N21amb)-His
FEATURES             Location/Qualifiers
     source          1..5788
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      36..491
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(123..142)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     333..354
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(583..1398)
                     /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(583..1398)
                     /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     1306..1325
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1390..1409
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1520..2108
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2009..2028
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2262..2279
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2294..2433
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2359..2378)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2519..2541
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2535..2726)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3535..4617)
                     /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     4579..4598
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4618..4695)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4901..4920
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5004..5023
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5004..5022
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5023..5047
                     /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             5062..5084
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5647..5664
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5726..5744)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5740..5787
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
       61 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
      121 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
      181 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
      241 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
      301 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
      361 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
      421 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
      481 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
      541 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
      601 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
      661 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
      721 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
      781 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
      841 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
      901 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
      961 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     1021 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     1081 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     1141 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     1201 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     1261 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     1321 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     1381 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     1441 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1501 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1561 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1621 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     1681 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1741 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1801 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1861 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1921 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1981 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     2041 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     2101 tatggaaaaa cgccagcaac gcggcctttt tacggttccc ggccttttgc tggccttttg
     2161 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     2221 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     2281 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2341 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     2401 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     2461 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     2521 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     2581 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     2641 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     2701 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     2761 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     2821 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     2881 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     2941 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     3001 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     3061 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     3121 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     3181 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     3241 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     3301 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     3361 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     3421 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     3481 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     3541 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3601 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     3661 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     3721 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     3781 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     3841 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     3901 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     3961 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     4021 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     4081 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     4141 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     4201 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     4261 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     4321 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     4381 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     4441 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     4501 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     4561 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     4621 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     4681 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     4741 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     4801 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     4861 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     4921 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     4981 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
     5041 caattcccct gtagaaataa ttttgtttaa ctttaagaag gagatataca tatgcaggat
     5101 agcaccagcg atctgattcc ggcaccgcct ctgagcaaag ttccgctgca gcagtagttt
     5161 caggataatc agtttcacgg caaatggtat gttgttggtc tggcaggtaa tcgtattctg
     5221 cgtgatgatc agcatccgat gaatatgtat gccaccatct atgaactgaa agaggacaaa
     5281 agctataacg tgaccagcgt tattagcagc cacaaaaaat gcgaatatac cattgcaacc
     5341 tttgttccgg gtagccagcc tggtgaattt accctgggta acattaaaag ctatggcgat
     5401 aaaaccagct atctggttcg tgttgttagc accgattata accagtatgc cgtggttttt
     5461 ttcaaactgg ccgaagataa tgccgaattt tttgccatta ccatttacgg tcgtaccaaa
     5521 gaactggcaa gcgagctgaa agaaaacttt attcgtttta gcaaaagcct gggtctgccg
     5581 gaaaatcata ttgtgtttcc ggttccgatt gatcagtgta ttgatggtgg tagcggtagc
     5641 ggttcacatc atcatcacca tcattaactc gagtaagatc cggctgctaa caaagcccga
     5701 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     5761 tctaaacggg tcttgagggg ttttttgc
//
