LOCUS       sequence-334767-        6484 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CTLA-4 with FLN fingerprint domain, ELP linker, 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 6484)
  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 6484)
  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-CTLA4-FLN-ELP-His-ybbr
FEATURES             Location/Qualifiers
     source          1..6484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      8..463
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(95..114)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     305..326
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(555..1370)
                     /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(555..1370)
                     /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     1278..1297
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1362..1381
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1492..2080
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1981..2000
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2234..2251
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2266..2405
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2331..2350)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2491..2513
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2507..2698)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3507..4589)
                     /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     4551..4570
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4590..4667)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4873..4892
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4976..4995
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4976..4994
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4995..5019
                     /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             5034..5056
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6276..6293
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6394..6412)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6408..6455
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
       61 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      121 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      181 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
      241 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
      301 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
      361 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
      421 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
      481 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      541 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
      601 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
      661 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
      721 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
      781 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
      841 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
      901 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
      961 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     1021 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     1081 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     1141 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     1201 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     1261 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     1321 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     1381 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga
     1441 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     1501 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     1561 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     1621 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     1681 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     1741 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     1801 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     1861 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     1921 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1981 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2041 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     2101 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     2161 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     2221 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     2281 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     2341 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     2401 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     2461 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     2521 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     2581 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     2641 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     2701 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     2761 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     2821 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     2881 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     2941 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     3001 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     3061 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     3121 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     3181 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     3241 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     3301 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     3361 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     3421 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     3481 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3541 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     3601 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     3661 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     3721 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     3781 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     3841 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     3901 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     3961 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     4021 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     4081 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     4141 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     4201 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     4261 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     4321 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     4381 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     4441 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     4501 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     4561 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     4621 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     4681 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     4741 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     4801 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     4861 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     4921 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     4981 cgactcacta taggggaatt gtgagcggat aacaattccc ctgtagaaat aattttgttt
     5041 aactttaaga aggagatata catatgcatg ttgcacagcc tgcagttgtt ctggcaagca
     5101 gccgtggtat tgcaagcttt gtttgtgaat atgcaagtcc gggtaaagca accgaagttc
     5161 gtgttaccgt tctgcgtcag gcagatagcc aggttaccga agtttgtgca gcaacctata
     5221 tgatgggtaa tgaactgacc tttctggatg atagcatttg taccggcacc agcagcggta
     5281 atcaggttaa tctgaccatt cagggtctgc gtgcaatgga taccggtctg tatatttgta
     5341 aagtcgagct gatgtatccg cctccgtatt atcttggtat tggtaatggc acccagatct
     5401 atgttattga tccggaacct ggtagcggta gcggttcagg tagcgctgac ccggaaaaat
     5461 cttacgctga aggtccgggt ctggacggtg gtgaatcctt ccagccgtct aaattcaaaa
     5521 tccacgctgt tgacccggac ggtgttcacc gtaccgacgg tggtgacggt ttcgttgtta
     5581 ccatcgaagg tccggctccg gttgacccgg ttatggttga caacggtgac ggtacctacg
     5641 acgttgaatt cgaaccgaaa gaagctggtg actacgttat caacctgacc ctggacggtg
     5701 acaacgttaa cggtttcccg aaaaccgtta ccgttaaacc ggctccgggc tccggttctg
     5761 gttcccacgg cgtgggtgtt ccaggtatgg gtgtgcctgg tgttggtgtt cctggcgttg
     5821 gcgtgccagg tgtgggagta ccaggcgtag gcgttcccgg tgtaggcgtg cctggcgtcg
     5881 gtgtacccgg tgtcggagtc cctggtgaag gtgtgccagg cgaaggtgtg ccgggcgtgg
     5941 gtgttccagg tatgggtgtg cctggtgttg gtgttcctgg cgttggcgtg ccaggtgtgg
     6001 gagtaccagg cgtaggcgtt cccggtgtag gcgtgcctgg cgtcggtgta cccggtgtcg
     6061 gagtccctgg tgaaggtgtg ccaggcgaag gtgtgccggg cgtgggtgtt ccaggtatgg
     6121 gtgtgcctgg tgttggtgtt cctggcgttg gcgtgccagg tgtgggagta ccaggcgtag
     6181 gcgttcccgg tgtaggcgtg cctggcgtcg gtgtacccgg tgtcggagtc cctggtgaag
     6241 gtgtgccagg cgaaggtgtg ccgggctggc gcggtcatca tcatcaccat catggtagcg
     6301 actctctgga attcatcgct tctaaactgg cttaactcga gtaagatccg gctgctaaca
     6361 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     6421 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     6481 tggc
//
