LOCUS       sequence-302475-        6805 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of cell-surface protein extracellular domain 
            fused to Fc(DAPA)-Avi-6xHis. Protein is secreted from cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6805)
  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 6805)
  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-BTN2A2-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6805
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     124..143
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     149..168
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      254..842
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     743..762
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     996..1013
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1111..1128
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1112..1128
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1584..1600
                     /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)."
     enhancer        1767..2146
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2147..2350
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2301..2321
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2347..2371
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        2581..2599
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2581..2598
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     2612..2631
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             4147..4191
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4192..4209
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4349..4366)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4355..4466
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4584..4604)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4589..4918
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4769..4904
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4831..4850
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4966..5013
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5032..5631
                     /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(5032..5051)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5528..5548
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5761..5882
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5798..5817)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5852..5871
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5935..6726,6727..6795))
                     /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     6558..6577
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6796..6805,1..82))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
       61 tgtatttaga aaaataaaca aataggggtc agtgttacaa ccaattaacc aattctgaac
      121 attatcgcga gcccatttat acctgaatat ggctcataac accccttgct catgaccaaa
      181 atcccttaac gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga
      241 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
      301 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
      361 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
      421 tagcccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
      481 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      541 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      601 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
      661 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      721 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      781 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      841 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      901 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
      961 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     1021 aaggcgagag tagggaactg ccaggcatca aactaagcag aaggcccctg acggatggcc
     1081 tttttgcgtt tctacaaact ctttctgtgt tgtaaaacga cggccagtct taagctcggg
     1141 ccccctgggc ggttctgata acgagtaatc gttaatccgc aaataacgta aaaacccgct
     1201 tcggcgggtt tttttatggg gggagtttag ggaaagagca tttgtcagaa tatttaaggg
     1261 cgcctgtcac tttgcttgat atatgagaat tatttaacct tataaatgag aaaaaagcaa
     1321 cgcactttaa ataagatacg ttgctttttc gattgatgaa cacctataat taaactattc
     1381 atctattatt tatgattttt tgtatataca atatttctag tttgttaaag agaattaaga
     1441 aaataaatct cgaaaataat aaagggaaaa tcagtttttg atatcaaaat tatacatgtc
     1501 aacgataata caaaatataa tacaaactat aagatgttat cagtatttat tatcatttag
     1561 aataaatttt gtgtcgccct taattgtgag cggataacaa ttacgagctt catgcacagt
     1621 ggcgttgact caatattggc cattagccat attattcatt ggttatatag cataaatcaa
     1681 tattggctat tggccattgc atacgttgta tctatatcat aatatgtaca tttatattgg
     1741 ctcatgtcca atatgaccgc catgttggca ttgattattg actagttatt aatagtaatc
     1801 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     1861 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     1921 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     1981 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     2041 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     2101 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     2161 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     2221 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     2281 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     2341 aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
     2401 cctccataga agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac
     2461 gcggattccc cgtgccaaga gtgacgtaag taccgcctat agagtctata ggcccacccc
     2521 cttggcttcg ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg
     2581 atttaggtga cactatagaa taacatccac tttgcctttc tctccacagg tgtccactcc
     2641 caggtccaac tgcacctcgg ttctatcgaa aacgcgcctg ctagcgccac catgaagacc
     2701 atcattgccc tgagctacat cttctgcctg gtgttcgctg cggccgccca gtttacagtt
     2761 gtgggacctg ccaatcctat cctggccatg gtcggagaga acaccacact gagatgccat
     2821 ctgagccccg agaagaacgc cgaggatatg gaagtgcggt ggttcagaag ccagttcagc
     2881 cctgccgtgt tcgtgtacaa aggcggcaga gaacggaccg aggaacagat ggaagagtac
     2941 cggggcagaa tcaccttcgt gtccaaggac atcaacagag gcagcgtggc cctggtcatc
     3001 cacaatgtga cagcccaaga gaacggcatc taccggtgct acttccaaga gggcagaagc
     3061 tacgacgagg ccatcctgag actggtggtt gccggactgg gaagcaagcc cctgatcgag
     3121 attaaggccc aagaggatgg cagcatctgg ctggaatgta tcagcggcgg atggtatcct
     3181 gagcctctga ccgtttggag agatccctat ggcgaagtgg tgcccgctct gaaagaagtg
     3241 tctatcgccg acgccgacgg cctgtttatg gtcacaacag ccgtgatcat ccgggacaaa
     3301 tacgtgcgga atgtgtcctg cagcgtgaac aacacactgc tgggccaaga gaaagaaacc
     3361 gtgatcttca tccccgagag cttcatgcct agcgcctctc catggatggt ggctctggct
     3421 gtgattctga ccgcttctcc ctggatgggc gcgccgggcg gcgacaagac ccacacctgc
     3481 cccccctgcc cagctccaga actgctggga gggccttccg tgttcctgtt cccccccaag
     3541 cccaaggaca ccctgatgat cagcaggacc cccgaggtga cctgcgtggt ggtggccgtg
     3601 tcccacgagg acccagaggt gaagttcaac tggtacgtgg acggcgtgga ggtgcacaac
     3661 gccaagacca agcccagaga ggagcagtac aacagcacct acagggtggt gtccgtgctg
     3721 accgtgctgc accaggactg gctgaacggc aaagaataca agtgcaaagt ctccaacaag
     3781 gccctggctg ccccaatcga aaagacaatc agcaaggcca agggccagcc acgggagccc
     3841 caggtgtaca ccctgccccc cagccgggag gagatgacca agaaccaggt gtccctgacc
     3901 tgtctggtga agggcttcta ccccagcgat atcgccgtgg agtgggagag caacggccag
     3961 cccgagaaca actacaagac caccccccca gtgctggaca gcgacggcag cttcttcctg
     4021 tacagcaagc tgaccgtgga caagtccagg tggcagcagg gcaacgtgtt cagctgcagc
     4081 gtgatgcacg aggccctgca caaccactac acccagaagt ccctgagcct gagccccggc
     4141 ggcagcggtc ttaatgatat tttcgaggct cagaagattg agtggcatga acaccaccac
     4201 catcaccact aaggccggcc tgtagtcact ggcacaacgc gttacccggt aagccaatcg
     4261 ggtatacacg gtcgtcatac tgcagacagg gttcttctac tttgcaagat agtcttgagt
     4321 agtaaaataa atagatagag aaaatcagcc tcgactgtgc cttctagttg ccagccatct
     4381 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     4441 tcctaataaa atgaggaaat tgcatcacaa cactcaaccc tatctcggtc tattcttttg
     4501 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4561 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     4621 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     4681 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4741 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     4801 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     4861 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     4921 gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca
     4981 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catgaccgag
     5041 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     5101 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     5161 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     5221 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     5281 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     5341 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     5401 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     5461 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacatcc
     5521 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     5581 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acacgtgcta
     5641 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     5701 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     5761 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     5821 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     5881 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcattacca
     5941 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6001 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagcgc
     6061 tgcgatgata ccgcgagaac cacgctcacc ggctccggat ttatcagcaa taaaccagcc
     6121 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6181 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6241 tgccatcgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6301 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6361 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6421 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6481 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6541 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6601 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6661 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6721 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6781 atgttgaata ctcatattct tcctt
//
