LOCUS       sequence-337388-        5788 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses anticalin targeting CTLA-4 with the codon encoding E143 
            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 Apr 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 (E143amb)-His
FEATURES             Location/Qualifiers
     source          1..5788
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(78..96)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      92..139
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      176..631
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(263..282)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     473..494
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(723..1538)
                     /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(723..1538)
                     /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     1446..1465
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1530..1549
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1660..2248
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2149..2168
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2402..2419
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2434..2573
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2499..2518)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2659..2681
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2675..2866)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3675..4757)
                     /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     4719..4738
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4758..4835)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5041..5060
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5144..5163
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5144..5162
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5163..5187
                     /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             5202..5224
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(5787..5788,1..16)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 tcatcatcac catcattaac tcgagtaaga tccggctgct aacaaagccc gaaaggaagc
       61 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
      121 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
      181 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
      241 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
      301 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
      361 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
      421 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
      481 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
      541 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
      601 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
      661 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
      721 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
      781 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
      841 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
      901 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
      961 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     1021 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     1081 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     1141 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     1201 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     1261 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     1321 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     1381 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     1441 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     1501 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     1561 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     1621 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1681 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1741 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1801 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1861 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1921 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1981 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2041 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2101 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2161 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2221 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttccc
     2281 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2341 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2401 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2461 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     2521 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     2581 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2641 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     2701 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     2761 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     2821 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     2881 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     2941 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     3001 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     3061 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     3121 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     3181 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     3241 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     3301 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     3361 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     3421 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     3481 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     3541 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     3601 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     3661 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3721 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     3781 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     3841 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     3901 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     3961 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     4021 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     4081 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     4141 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     4201 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     4261 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     4321 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     4381 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     4441 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     4501 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     4561 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     4621 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     4681 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     4741 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     4801 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     4861 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     4921 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     4981 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     5041 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     5101 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     5161 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaagaag
     5221 gagatataca tatgcaggat agcaccagcg atctgattcc ggcaccgcct ctgagcaaag
     5281 ttccgctgca gcagaatttt caggataatc agtttcacgg caaatggtat gttgttggtc
     5341 tggcaggtaa tcgtattctg cgtgatgatc agcatccgat gaatatgtat gccaccatct
     5401 atgaactgaa agaggacaaa agctataacg tgaccagcgt tattagcagc cacaaaaaat
     5461 gcgaatatac cattgcaacc tttgttccgg gtagccagcc tggtgaattt accctgggta
     5521 acattaaaag ctatggcgat aaaaccagct atctggttcg tgttgttagc accgattata
     5581 accagtatgc cgtggttttt ttcaaactgg ccgaagataa tgccgaattt tttgccatta
     5641 ccatttacgg tcgtaccaaa tagctggcaa gcgagctgaa agaaaacttt attcgtttta
     5701 gcaaaagcct gggtctgccg gaaaatcata ttgtgtttcc ggttccgatt gatcagtgta
     5761 ttgatggtgg tagcggtagc ggttcaca
//
