LOCUS       sequence-400992-        8419 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of cell-surface protein extracellular domain 
            fused to COMP5AP-AviTag-9xHis. Protein is secreted from cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8419)
  AUTHORS   Wojtowicz WM, Vielmetter J, Fernandes RA, Siepe DH, Eastman CL, 
            Chisholm GB, Cox S, Klock H, Anderson PW, Rue SM, Miller JJ, Glaser 
            SM, Bragstad ML, Vance J, Lam AW, Lesley SA, Zinn K, Garcia KC
  TITLE     A Human IgSF Cell-Surface Interactome Reveals a Complex Network of 
            Protein-Protein Interactions.
  JOURNAL   Cell. 2020 Aug 20;182(4):1027-1043.e17. doi: 
            10.1016/j.cell.2020.07.025.
  PUBMED    32822567
REFERENCE   2  (bases 1 to 8419)
  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: 
            "10.1016/j.cell.2020.07"; journalName: "Cell"; date: "2020-08-20- 
            20"; volume: "182"; issue: "4"; pages: "1027-1043.e17"
COMMENT     Sequence Label: pD649-HAsp-LAG3-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     815..832
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             2258..2293
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             2303..2326
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3986..4030
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4031..4057
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(4197..4214)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4203..4314
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4432..4452)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4437..4766
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4617..4752
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4679..4698
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4814..4861
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4880..5479
                     /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(4880..4899)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5376..5396
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5609..5730
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5646..5665)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5700..5719
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5783..6574,6575..6643))
                     /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     6406..6425
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6644..6735)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6777..6796
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6802..6821
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6907..7495
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7396..7415
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7649..7666
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7764..7781
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7765..7781
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8237..8253
                     /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)."
ORIGIN
        1 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
      901 cgaaaacgcg cctgctagcg ccaccatgaa gaccatcatt gccctgagct acatcttctg
      961 cctggtgttc gctgcggccg ccgttccagt tgtttgggct caagaaggtg cccctgctca
     1021 gctgccttgc agccctacaa tcccactgca ggatctgagc ctgcttcgga gagctggcgt
     1081 gacatggcag caccagcctg attctggacc tcctgctgct gctcctggac atccacttgc
     1141 tccaggacct catcctgccg ctcctagttc ttggggccct agacctagac ggtacaccgt
     1201 gctgtctgtt ggacctggcg gactgagatc tggcagactg cctctgcaac ccagagtgca
     1261 gctggacgaa agaggcagac agaggggcga tttcagcctg tggcttagac ctgccagaag
     1321 ggctgacgcc ggcgagtata gagctgccgt gcacctgaga gatagagccc tgagctgccg
     1381 gctgagactg agactgggac aagccagcat gaccgcctct ccacctggat ctctgagagc
     1441 cagcgattgg gtcatcctga actgcagctt ctcccggcct gatagaccag ccagcgtgca
     1501 ctggttcaga aacagaggcc agggcagagt gcccgtgcgg gaatctcctc atcatcacct
     1561 ggccgagagc tttctgttcc tgcctcaagt gtcccctatg gactctggcc cttggggctg
     1621 tatcctgacc tacagagatg gcttcaacgt gtccatcatg tacaacctga ccgtgctggg
     1681 cctcgagcct cctacacctc tgacagttta tgccggcgct ggcagcagag tgggcctgcc
     1741 ttgtagactt cctgccggcg tgggcaccag aagctttctg acagccaagt ggacacctcc
     1801 tggcggcgga cctgatttgc ttgtgacagg cgacaacggc gacttcaccc tgagactcga
     1861 ggatgtgtct caggcccagg ccggcacata cacctgtcac atccatctgc aagaacagca
     1921 gctgaacgcc accgtgacac tggccatcat cacagtgacc cctaagagct ttggcagccc
     1981 tggcagcctg ggaaagctgc tgtgtgaagt gacacccgtg tccggccaag agcgctttgt
     2041 ttggagcagc ctggataccc ctagccagag aagctttagc ggcccttggc tggaagccca
     2101 agaagcccag ctgctttctc agccttggca gtgccagctg taccagggcg aaagactgct
     2161 gggagccgcc gtgtacttta ccgagcttag ttctcctggc gctcaaagaa gcggcagagc
     2221 acctggtgca ttgcctgccg gacatcttgg cgcgccggaa gatcaagttg atccaagatt
     2281 gattgatggc aagggcggat ctctggaggt gctgttccag ggaccaggcg gaagcggcaa
     2341 tcccggcggc gatctggccc cccagatgct gcgcgagctg caggagacca atgccgccct
     2401 gcaggacgtg cgcgagctgc tgcgccaccg cgtgaaggag atcaccttcc tgaagaacac
     2461 cgtgatggag tgcgacgcct gcggtatgca gccctctaga ggctccggct ccatcatccc
     2521 agttgaggag gagaacccgg acttctggaa ccgcgaggca gccgaggccc tgggtgccgc
     2581 caagaagctg cagcctgcac agacagccgc caagaacctc atcatcttcc tgggcgatgg
     2641 gatgggggtg tctacggtga cagctgccag gatcctaaaa gggcagaaga aggacaaact
     2701 ggggcctgag atacccctgg ccatggaccg cttcccatat gtggctctgt ccaagacata
     2761 caatgtagac aaacatgtgc cagacagtgg agccacagcc acggcctacc tgtgcggggt
     2821 caagggcaac ttccagacca ttggcttgag tgcagccgcc cgctttaacc agtgcaacac
     2881 gacacgcggc aacgaggtca tctccgtgat gaatcgggcc aagaaagcag ggaagtcagt
     2941 gggagtggta accaccacac gagtgcagca cgcctcgcca gccggcacct acgcccacac
     3001 ggtgaaccgc aactggtact cggacgccga cgtgcctgcc tcggcccgcc aggaggggtg
     3061 ccaggacatc gctacgcagc tcatctccaa catggacatt gacgtgatcc taggtggagg
     3121 ccgaaagtac atgtttcgca tgggaacccc agaccctgag tacccagatg actacagcca
     3181 aggtgggacc aggctggacg ggaagaatct ggtgcaggaa tggctggcga agcgccaggg
     3241 tgcccggtat gtgtggaacc gcactgagct catgcaggct tccctggacc cgtctgtgac
     3301 ccatctcatg ggtctctttg agcctggaga catgaaatac gagatccacc gagactccac
     3361 actggacccc tccctgatgg agatgacaga ggctgccctg cgcctgctga gcaggaaccc
     3421 ccgcggcttc ttcctcttcg tggagggtgg tcgcatcgac catggtcatc atgaaagcag
     3481 ggcttaccgg gcactgactg agacgatcat gttcgacgac gccattgaga gggcgggcca
     3541 gctcaccagc gaggaggaca cgctgagcct cgtcactgcc gaccactccc acgtcttctc
     3601 cttcggaggc taccccctgc gagggagctc catcttcggg ctggcccctg gcaaggcccg
     3661 ggacaggaag gcctacacgg tcctcctata cggaaacggt ccaggctatg tgctcaagga
     3721 cggcgcccgg ccggatgtta ccgagagcga gagcgggagc cccgagtatc ggcagcagtc
     3781 agcagtgccc ctggacgaag agacccacgc aggcgaggac gtggcggtgt tcgcgcgcgg
     3841 cccgcaggcg cacctggttc acggcgtgca ggagcagacc ttcatagcgc acgtcatggc
     3901 cttcgccgcc tgcctggagc cctacaccgc ctgcgacctg gcgccccccg ccggcaccac
     3961 cgacgccgcg cacccgggtg gcggaggact gaacgacatc ttcgaggctc agaaaatcga
     4021 atggcacgaa catcaccatc accatcacca tcaccattaa ggccggcctg tagtcactgg
     4081 cacaacgcgt tacccggtaa gccaatcggg tatacacggt cgtcatactg cagacagggt
     4141 tcttctactt tgcaagatag tcttgagtag taaaataaat agatagagaa aatcagcctc
     4201 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     4261 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcacaaca
     4321 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4381 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     4441 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4501 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     4561 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     4621 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4681 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     4741 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     4801 ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
     4861 caaggtgagg aactaaacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     4921 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     4981 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     5041 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     5101 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     5161 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     5221 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca
     5281 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     5341 cggggtgccc gccttcctgg agacatccgc gccccgcaac ctccccttct acgagcggct
     5401 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     5461 ccgcaagccc ggtgcctgac acgtgctacg agatttcgat tccaccgccg ccttctatga
     5521 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     5581 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     5641 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     5701 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     5761 gagcttggcg taatcatggt cattaccaat gcttaatcag tgaggcacct atctcagcga
     5821 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5881 gggagggctt accatctggc cccagcgctg cgatgatacc gcgagaacca cgctcaccgg
     5941 ctccggattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6001 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6061 cgccagttaa tagtttgcgc aacgttgttg ccatcgctac aggcatcgtg gtgtcacgct
     6121 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6181 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6241 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6301 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6361 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6421 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6481 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6541 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6601 caaaaaaggg aataagggcg acacggaaat gttgaatact catattcttc ctttttcaat
     6661 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6721 agaaaaataa acaaataggg gtcagtgtta caaccaatta accaattctg aacattatcg
     6781 cgagcccatt tatacctgaa tatggctcat aacacccctt gctcatgacc aaaatccctt
     6841 aacgtgagtt acgcgcgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6901 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6961 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     7021 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttagccca
     7081 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     7141 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     7201 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     7261 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     7321 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     7381 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7441 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     7501 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     7561 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     7621 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaaggcga
     7681 gagtagggaa ctgccaggca tcaaactaag cagaaggccc ctgacggatg gcctttttgc
     7741 gtttctacaa actctttctg tgttgtaaaa cgacggccag tcttaagctc gggccccctg
     7801 ggcggttctg ataacgagta atcgttaatc cgcaaataac gtaaaaaccc gcttcggcgg
     7861 gtttttttat ggggggagtt tagggaaaga gcatttgtca gaatatttaa gggcgcctgt
     7921 cactttgctt gatatatgag aattatttaa ccttataaat gagaaaaaag caacgcactt
     7981 taaataagat acgttgcttt ttcgattgat gaacacctat aattaaacta ttcatctatt
     8041 atttatgatt ttttgtatat acaatatttc tagtttgtta aagagaatta agaaaataaa
     8101 tctcgaaaat aataaaggga aaatcagttt ttgatatcaa aattatacat gtcaacgata
     8161 atacaaaata taatacaaac tataagatgt tatcagtatt tattatcatt tagaataaat
     8221 tttgtgtcgc ccttaattgt gagcggataa caattacgag cttcatgcac agtggcgttg
     8281 actcaatatt ggccattagc catattattc attggttata tagcataaat caatattggc
     8341 tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat tggctcatgt
     8401 ccaatatgac cgccatgtt
//
