LOCUS       sequence-396615-        7420 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 7420)
  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 7420)
  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-CD276-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..7420
                     /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             2996..3040
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             3041..3058
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3198..3215)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3204..3315
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3433..3453)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3438..3767
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3618..3753
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3680..3699
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3815..3862
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3881..4480
                     /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(3881..3900)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4377..4397
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4610..4731
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4647..4666)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4701..4720
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(4784..5575,5576..5644))
                     /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     5407..5426
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5645..5736)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5778..5797
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5803..5822
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5908..6496
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6397..6416
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6650..6667
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6765..6782
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6766..6782
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7238..7254
                     /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 ccttggaagt gcaagtgcct gaagatcctg tggtggccct
     1021 cgtgggaaca gatgccacac tgtgctgtag ctttagcccc gagcctggat tttctctggc
     1081 ccagctgaat ctgatctggc agctgaccga taccaagcag ctggtgcact ctttcgccga
     1141 aggccaggat cagggcagcg cctatgctaa cagaaccgct ctgtttcccg atctgctggc
     1201 ccagggaaat gccagcctga gactgcagag agtcagagtt gccgacgagg gcagcttcac
     1261 ctgtttcgtg tccatcagag atttcggctc tgccgccgtg tctctgcaag tggccgctcc
     1321 ttacagcaag cccagcatga ccctggaacc taacaaggac ctgaggcctg gcgacaccgt
     1381 gaccatcaca tgtagcagct accagggcta tcccgaggcc gaagtgtttt ggcaggatgg
     1441 acaaggcgtg cccctgaccg gcaatgtgac aacatctcag atggccaacg agcagggcct
     1501 gttcgatgtg cactctatcc tgagagtggt gctgggcgcc aatggcacat atagctgtct
     1561 cgtgcggaac cccgttctgc agcaggatgc ccacagcagc gtgacaatca caccccagag
     1621 atctccaaca ggcgccgtgg aagttcaggt gccagaggac cctgttgtgg ctcttgtggg
     1681 caccgatgct accctgagat gctcttttag ccctgaacca ggcttcagcc tcgctcagct
     1741 gaacctcatt tggcaactca cagacacaaa acagctcgtc cactccttca ccgagggaag
     1801 agatcagggc tccgcctacg ccaataggac agcactgttt ccagacctgc tcgctcaagg
     1861 caacgctagt ctgaggctgc aacgcgttag agtggccgat gaaggctcct ttacctgctt
     1921 cgtcagcatc cgcgattttg gcagtgctgc cgtcagcctg caagtcgctg ccccttactc
     1981 caagccttct atgaccctcg agccgaacaa agatctcaga cccggcgata cagtgaccat
     2041 tacctgcagc tcctacagag gctacccaga ggctgaggtt ttctggcaag acggacaggg
     2101 cgttccactg actggaaacg tgaccaccag ccagatggct aatgaacagg gactctttga
     2161 cgtgcacagc gtgctgagag tcgttctggg agctaacggc acctattctt gcctcgtcag
     2221 aaaccctgtg ctccagcagg acgctcacgg ctctgtgaca attaccggcc agcctatgac
     2281 ctttccacct gaggctggcg cgccgggcgg cgacaagacc cacacctgcc ccccctgccc
     2341 agctccagaa ctgctgggag ggccttccgt gttcctgttc ccccccaagc ccaaggacac
     2401 cctgatgatc agcaggaccc ccgaggtgac ctgcgtggtg gtggccgtgt cccacgagga
     2461 cccagaggtg aagttcaact ggtacgtgga cggcgtggag gtgcacaacg ccaagaccaa
     2521 gcccagagag gagcagtaca acagcaccta cagggtggtg tccgtgctga ccgtgctgca
     2581 ccaggactgg ctgaacggca aagaatacaa gtgcaaagtc tccaacaagg ccctggctgc
     2641 cccaatcgaa aagacaatca gcaaggccaa gggccagcca cgggagcccc aggtgtacac
     2701 cctgcccccc agccgggagg agatgaccaa gaaccaggtg tccctgacct gtctggtgaa
     2761 gggcttctac cccagcgata tcgccgtgga gtgggagagc aacggccagc ccgagaacaa
     2821 ctacaagacc acccccccag tgctggacag cgacggcagc ttcttcctgt acagcaagct
     2881 gaccgtggac aagtccaggt ggcagcaggg caacgtgttc agctgcagcg tgatgcacga
     2941 ggccctgcac aaccactaca cccagaagtc cctgagcctg agccccggcg gcagcggtct
     3001 taatgatatt ttcgaggctc agaagattga gtggcatgaa caccaccacc atcaccacta
     3061 aggccggcct gtagtcactg gcacaacgcg ttacccggta agccaatcgg gtatacacgg
     3121 tcgtcatact gcagacaggg ttcttctact ttgcaagata gtcttgagta gtaaaataaa
     3181 tagatagaga aaatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     3241 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     3301 tgaggaaatt gcatcacaac actcaaccct atctcggtct attcttttga tttataaggg
     3361 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3421 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3481 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3541 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3601 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3661 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3721 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3781 cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta
     3841 tatcggcata gtataatacg acaaggtgag gaactaaacc atgaccgagt acaagcccac
     3901 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     3961 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     4021 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     4081 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     4141 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     4201 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     4261 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     4321 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacatccg cgccccgcaa
     4381 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     4441 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cacgtgctac gagatttcga
     4501 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4561 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4621 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4681 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4741 tataccgtcg acctctagct agagcttggc gtaatcatgg tcattaccaa tgcttaatca
     4801 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     4861 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagcgct gcgatgatac
     4921 cgcgagaacc acgctcaccg gctccggatt tatcagcaat aaaccagcca gccggaaggg
     4981 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5041 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccatcgcta
     5101 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5161 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5221 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5281 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5341 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5401 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5461 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5521 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5581 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     5641 tcatattctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     5701 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggtcagtgtt acaaccaatt
     5761 aaccaattct gaacattatc gcgagcccat ttatacctga atatggctca taacacccct
     5821 tgctcatgac caaaatccct taacgtgagt tacgcgcgcg tcgttccact gagcgtcaga
     5881 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5941 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6001 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     6061 agtgtagccg tagttagccc accacttcaa gaactctgta gcaccgccta catacctcgc
     6121 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6181 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6241 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6301 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6361 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6421 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6481 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6541 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     6601 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     6661 gagcgaggaa gcggaaggcg agagtaggga actgccaggc atcaaactaa gcagaaggcc
     6721 cctgacggat ggcctttttg cgtttctaca aactctttct gtgttgtaaa acgacggcca
     6781 gtcttaagct cgggccccct gggcggttct gataacgagt aatcgttaat ccgcaaataa
     6841 cgtaaaaacc cgcttcggcg ggttttttta tggggggagt ttagggaaag agcatttgtc
     6901 agaatattta agggcgcctg tcactttgct tgatatatga gaattattta accttataaa
     6961 tgagaaaaaa gcaacgcact ttaaataaga tacgttgctt tttcgattga tgaacaccta
     7021 taattaaact attcatctat tatttatgat tttttgtata tacaatattt ctagtttgtt
     7081 aaagagaatt aagaaaataa atctcgaaaa taataaaggg aaaatcagtt tttgatatca
     7141 aaattataca tgtcaacgat aatacaaaat ataatacaaa ctataagatg ttatcagtat
     7201 ttattatcat ttagaataaa ttttgtgtcg cccttaattg tgagcggata acaattacga
     7261 gcttcatgca cagtggcgtt gactcaatat tggccattag ccatattatt cattggttat
     7321 atagcataaa tcaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg
     7381 tacatttata ttggctcatg tccaatatga ccgccatgtt
//
