LOCUS       sequence-334766-        5788 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses anticalin targeting CTLA-4 with the codon encoding E60 
            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 (E60amb)-His
FEATURES             Location/Qualifiers
     source          1..5788
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      18..473
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(105..124)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     315..336
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(565..1380)
                     /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(565..1380)
                     /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     1288..1307
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1372..1391
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1502..2090
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1991..2010
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2244..2261
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2276..2415
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2341..2360)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2501..2523
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2517..2708)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3517..4599)
                     /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
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLSASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4561..4580
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4600..4677)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4883..4902
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4986..5005
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4986..5004
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5005..5029
                     /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             5044..5066
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5629..5646
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5708..5726)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5722..5769
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
       61 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
      121 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
      181 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
      241 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
      301 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
      361 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
      421 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
      481 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
      541 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
      601 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
      661 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
      721 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
      781 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
      841 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
      901 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
      961 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     1021 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     1081 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     1141 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     1201 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     1261 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     1321 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     1381 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     1441 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     1501 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     1561 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     1621 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     1681 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     1741 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     1801 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     1861 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     1921 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     1981 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     2041 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     2101 acgcggcctt tttacggttc ccggcctttt gctggccttt tgctcacatg ttctttcctg
     2161 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     2221 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     2281 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     2341 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     2401 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     2461 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     2521 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     2581 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     2641 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     2701 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     2761 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     2821 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     2881 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     2941 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     3001 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     3061 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     3121 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     3181 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     3241 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     3301 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     3361 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     3421 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     3481 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3541 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3601 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     3661 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     3721 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     3781 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     3841 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     3901 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     3961 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     4021 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     4081 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     4141 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     4201 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     4261 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     4321 gctggcactc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     4381 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     4441 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     4501 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     4561 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     4621 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     4681 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     4741 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     4801 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     4861 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     4921 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     4981 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctgtagaaat
     5041 aattttgttt aactttaaga aggagatata catatgcagg atagcaccag cgatctgatt
     5101 ccggcaccgc ctctgagcaa agttccgctg cagcagaatt ttcaggataa tcagtttcac
     5161 ggcaaatggt atgttgttgg tctggcaggt aatcgtattc tgcgtgatga tcagcatccg
     5221 atgaatatgt atgccaccat ctatgaactg aaataggaca aaagctataa cgtgaccagc
     5281 gttattagca gccacaaaaa atgcgaatat accattgcaa cctttgttcc gggtagccag
     5341 cctggtgaat ttaccctggg taacattaaa agctatggcg ataaaaccag ctatctggtt
     5401 cgtgttgtta gcaccgatta taaccagtat gccgtggttt ttttcaaact ggccgaagat
     5461 aatgccgaat tttttgccat taccatttac ggtcgtacca aagaactggc aagcgagctg
     5521 aaagaaaact ttattcgttt tagcaaaagc ctgggtctgc cggaaaatca tattgtgttt
     5581 ccggttccga ttgatcagtg tattgatggt ggtagcggta gcggttcaca tcatcatcac
     5641 catcattaac tcgagtaaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     5701 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     5761 ggttttttgc tgaaaggagg aactatat
//
