LOCUS       sequence-488354-        9217 bp    DNA     circular SYN 03-OCT-2025
DEFINITION  Expression of ECD of WT- Trp1 fused to display TMD..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9217)
  TITLE     Pyroglutamation of cell surface proteins CD47 and TRP1 by glutaminyl
            cyclase modulates therapeutic antibody binding.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9217)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 3, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: Trp1-Ala-display
FEATURES             Location/Qualifiers
     source          1..9217
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1480..1518
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             1522..1551
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    1789..2340
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1933..1950)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             2343..2942
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2343..2362)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2839..2859
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2967..3556
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3021..3041)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3439..3450)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3643..3876
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     rep_origin      4137..4272
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     rep_origin      complement(4390..4978)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4470..4489)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(5340..5408,5409..6200)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5558..5577)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6213..6349
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     enhancer        6426..6729
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6730..6933
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6884..6904
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6948..7128
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7175..7300
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7793..8026
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8211..8255
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    8522..8639
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8735..9136
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
ORIGIN
        1 gccgccacca tgtccgcccc taaattgctg agcctcggat gcatattctt ccccctcctc
       61 ctttttcagc aggcccgggc cgccttcccg agacaatgcg ctactgtgga ggcccttaga
      121 tccggcatgt gctgcccgga cctctccccc gttagcggcc ccggaactga ccgctgcgga
      181 tctagtagcg gaagaggtag atgtgaggca gttacagctg atagtcgtcc tcactcacca
      241 cagtaccccc atgacggtcg ggacgatcgg gaagtctggc ctctgaggtt ttttaataga
      301 acttgtcact gtaacggcaa tttcagcggg cataactgtg ggacttgtag gcccggttgg
      361 cgtggcgctg cttgcgatca aagagtactt atcgtgcgga ggaacttatt ggatctgagc
      421 aaagaagaga agaaccactt cgtgcgcgca ctggatatgg ctaagagaac cacccatcca
      481 ctgttcgtca ttgccactcg aagatccgaa gagatccttg gcccagatgg gaacactcct
      541 cagtttgaaa acattagcat ttacaattac tttgtttgga cccattacta ctcagtaaaa
      601 aagactttcc tgggcgtcgg tcaggaatcc tttggagaag tggatttctc acatgaaggg
      661 ccagccttct taacatggca cagataccat ctgctccggc ttgaaaagga catgcaagag
      721 atgctccaag agccctcctt cagcctgccc tactggaact tcgccacagg gaagaatgtg
      781 tgcgatattt gtaccgacga tctgatgggc agtagaagta acttcgattc cacgctgatt
      841 tcaccaaact cagtcttttc ccagtggcgg gttgtgtgcg atagcctgga ggactacgac
      901 actctcggca cactctgcaa cagcacagag gacggcccca tcaggagaaa tcctgctggg
      961 aacgttgccc gacccatggt tcagcggtta cctgagcccc aggatgtggc ccagtgcctt
     1021 gaagtgggac tctttgacac tcccccattt tattccaata gcacaaactc cttccgcaac
     1081 acagtcgaag gctacagtga tcctacgggt aagtatgacc ctgccgttag gagtctccac
     1141 aacctggctc atctgttctt gaatggaacc ggaggccaga cgcaccttag tcctaacgac
     1201 cctattttcg ttcttttgca tactttcaca gacgcagtct tcgatgaatg gcttcgccga
     1261 tacaacgcag atatctcaac cttcccatta gaaaatgctc caattggcca caatagacag
     1321 tacaacatgg tccccttctg gcctcctgtc accaacactg aaatgtttgt gaccgccccc
     1381 gacaacctcg gatacaccta tgagatccag tggcccggcg ggggaggaag tggtggcggt
     1441 ggtagcggcg gtggtggctc cggcggtggt ggttcaccca gcagactcga ggaggagctg
     1501 cgacgacgcc tgactgagcc cgagcagaaa ctgatttcag aagaggacct gaacgctgtt
     1561 gggcaagaca cacaggaagt cattgtggtc ccgcattcat tgccgttcaa agtcgtggtt
     1621 atctccgcca ttctggcctt agtggttctt acaatcatta gtctcatcat tctgataatg
     1681 ctgtggcaga agaaaccccg ctaaactagt gtctcgagct tattccagat gcgtgcggat
     1741 ggaattcgag ctcggtacca tgccaaaagc aaagcgctat cgcgccttac gttactggcc
     1801 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc
     1861 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta
     1921 ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag
     1981 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
     2041 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg
     2101 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat
     2161 ggctcccctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta
     2221 tgggatctga tctggggcct cggtgcacat gcttttcatg tgtttagtcg aggttaaaaa
     2281 acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataata
     2341 acatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg
     2401 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg
     2461 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     2521 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     2581 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     2641 gttcccggct ggccgcgcag caacagatgg agggcctcct ggcgccgcac cggcccaagg
     2701 agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg
     2761 gcagcgccgt cgtgctcccc ggagtggagg ccgccgagcg cgccggggtg cccgccttcc
     2821 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     2881 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct
     2941 aggctagctt gactgactga gtcgacaatc aaccttttgg attacaaaat ttgtgaaaga
     3001 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     3061 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     3121 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     3181 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     3241 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     3301 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     3361 aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     3421 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     3481 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     3541 tgggccgcct ccccgcctgg aattcgagct cggtaccttt aagaccaatg acttacaagg
     3601 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     3661 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct
     3721 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     3781 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     3841 tcagaccctt ttagtcagtg tggaaaatct ctagcagtcc tggccaacgt gagcaccgtg
     3901 ctgacctcca aatatcgtta agctggagcc tgggagccgg cctggccctc cgcccccccc
     3961 acccccgcag cccacccctg gtctttgaat aaagtctgag tgagtggccg acagtgcccg
     4021 tggagttctc gtgacctgag gtgcagggcc ggcgctaggg acacgtccgt gcacgtgccg
     4081 aggccccctg tgcagctgca agggacaggc ctagccctgc aggcctaact ccgcccatcc
     4141 cgcccctaac tccgcccagt tccgcccatt ctccgcctca tggctgacta atttttttta
     4201 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggacgct
     4261 tttttggagg ccgaggcttt tgcaaagatc gaacaagaga caggacctgc aggttaatta
     4321 aatttaaatc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4381 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4441 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4501 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4561 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4621 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4681 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4741 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4801 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4861 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4921 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4981 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5041 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5101 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5161 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcatttaa
     5221 atggccggcc tggcgcgccg tttaaaccta gatattgata gtctgatcgg tcaacgtata
     5281 atcgagtcct agcttttgca aacatctatc aagagacagg atcagcagga ggctttcgca
     5341 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5401 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcrc
     5461 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5521 aagaacgctt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5581 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5641 ttgagtaytc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5701 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatyg
     5761 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5821 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5881 ctgtagcaat ggcaacaacs ttgcgyaaac tattaactgg cgaactactt actctagctt
     5941 cccggcaaca rttratagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6001 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6061 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6121 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6181 cactgattaa gcattggtaa ccgattctag gtgcattggc gcagaaaaaa atgcctgatg
     6241 cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc
     6301 aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag atcccgaatc
     6361 gtttaaacgc gatcgcagta atcaattacg gggtcattag ttcatagccc atatatggag
     6421 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     6481 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     6541 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     6601 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     6661 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     6721 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
     6781 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
     6841 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
     6901 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgggg tctctctggt
     6961 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     7021 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     7081 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
     7141 cagggacctg aaagcgaaag ggaaaccaga gnnnctctct cgacgcagga ctcggcttgc
     7201 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
     7261 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
     7321 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
     7381 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
     7441 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
     7501 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
     7561 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
     7621 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
     7681 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     7741 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     7801 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcstca atgacgctga
     7861 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     7921 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     7981 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     8041 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     8101 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     8161 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     8221 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     8281 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     8341 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     8401 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     8461 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcggttaa
     8521 cttttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
     8581 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttg
     8641 gctcccgatc gttgcgttac acacacaatt actgctgatc gagtgtagcc ttcgaatgaa
     8701 agaccccacc tgtaggtttg gcaagatagc tgcagtaacg ccattttgca aggcatggaa
     8761 aaataccaaa ccaagaatag agaagttcag atcaagggcg ggtacatgaa aatagctaac
     8821 gttgggccaa acaggatatc tgcggtgagc agtttcggcc ccggcccggg gccaagaaca
     8881 gatggtcacc gcagtttcgg ccccggcccg aggccaagaa cagatggtcc ccagatatgg
     8941 cccaaccctc agcagtttct taagacccat cagatgtttc caggctcccc caaggacctg
     9001 aaatgaccct gcgccttatt tgaattaacc aatcagcctg cttctcgctt ctgttcgcgc
     9061 gcttctgctt cccgagctct ataaaagagc tcacaacccc tcactcggcg cgccagtcct
     9121 ccgattgact gagtcgccct gatcattgtc gatcctacca tccactcgac acacccgcca
     9181 gggcccgcat ccaccatcgc agacttatca tggatcc
//
