LOCUS       sequence-303117-        8848 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 8848)
  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 8848)
  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-PRTG-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8848
                     /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             6190..6234
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6235..6252
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6392..6409)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6398..6509
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6627..6647)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6632..6961
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6812..6947
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6874..6893
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7009..7056
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7075..7674
                     /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(7075..7094)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7571..7591
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7804..7925
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7841..7860)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7895..7914
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7978..8769,8770..8838))
                     /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     8601..8620
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8839..8848,1..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 cggccgcctt tagcgagctg
     2761 agcttcgtga aagaacccca ggacgtgacc gtgaccagaa aggaccctgt ggtgctggat
     2821 tgtcaggccc acggcgaagt gcctatcaaa gtgacctggc tgaagaacgg cgccaagatg
     2881 agcgagaaca agcggatcga ggtgctgagc aacggcagcc tgtacatctc tgaggtggaa
     2941 ggcagaagag gcgagcagag cgacgagggc ttttaccagt gcctggccat gaacaaatac
     3001 ggcgccatcc tgagccagaa ggcccatctg gccctgagca caatctctgc ctttgaggtg
     3061 cagcccatca gcacagaagt gcacgaaggc ggagtggcca gattcgcctg caagatctct
     3121 tctcaccctc ctgccgtgat cacctgggag ttcaacagaa ccacactgcc catgaccatg
     3181 gaccggatca cagctttgcc aaccggcgtg ctgcagatct acgacgtgtc ccagagagac
     3241 agcggcaact acagatgtat cgccgccaca gtggcccaca gacggaagtc tatggaagcc
     3301 agcctgacag tgatccccgc caaagagagc aagagctttc acacccctac catcattgcc
     3361 ggacctcaga acatcaccac cagcctgcat cagaccgtgg tgctcgagtg tatggccacc
     3421 ggcaatccca agcctatcat cagctggtcc cggctggacc acaagagcat cgacgtgttc
     3481 aataccagag tgctcggcaa cggcaatctg atgatctccg atgtgcgact gcagcatgca
     3541 ggcgtgtacg tgtgtagagc caccacacct ggcaccagaa acttcaccgt ggccatggct
     3601 acactgacag tgctggcccc tcctagcttt gtggaatggc ctgagagcct gacaagacct
     3661 agagccggca ccgccagatt tgtgtgtcag gccgagggaa tcccatctcc taagatgagc
     3721 tggctgaaaa atggccggaa gatccacagc aacggccgga tcaagatgta caacagcaag
     3781 ctggtcatca accagatcat ccccgaggac gacgccatct atcagtgcat ggccgagaat
     3841 agccagggct ctatcctgag cagagccaga ctgaccgtgg tcatgagcga ggatagaccc
     3901 agcgctcctt acaacgtgca cgccgagaca atgagcagct ctgccattct gctggcctgg
     3961 gaaagacccc tgtacaactc cgacaaagtg atcgcctaca gcgtgcacta catgaaggcc
     4021 gaaggcctga acaacgagga ataccaggtg gtcatcggca acgacaccac acactacatc
     4081 atcgacgacc tggaacctgc cagcaactac accttctaca tcgtggccta catgcctatg
     4141 ggcgccagcc agatgtctga ccacgtgaca cagaacaccc tggaagatgt gcctctgcgg
     4201 cctcctgaga tcagcctgac tagcagaagc cccaccgaca tcctgatcag ctggctgcct
     4261 attcctgcca agtatcggag aggccaggtg gtgctgtaca gactgagctt cagactgtcc
     4321 accgagaaca gcatccaggt gctggaactg cctggcacaa cacacgagta cctgctggaa
     4381 ggactgaagc ccgattctgt gtacctcgtg cggattacag ccgccacaag agttggactg
     4441 ggagagtcta gcgtgtggac cagccataga acccctaagg ccacaagcgt gaaggcccct
     4501 aagtctcccg aactgcatct ggaacccctg aactgcacaa ccatctctgt gcggtggcag
     4561 caggatgtgg aagatacagc cgctatccag ggctacaagc tgtactacaa agaggaaggc
     4621 cagcaagaga acggccccat cttcctggat accaaggacc tgctgtacac cctgagcgga
     4681 ctggacccta gaagaaagta tcatgtgcgg ctgctggctt acaacaacat cgacgatggc
     4741 taccaggccg accagacagt gtctacacca ggatgcgtgt ccgtgcggga tagaatggtt
     4801 cctcctccac cacctccaca ccacctgtat gccaaggcca ataccagcag cagcatcttt
     4861 ctgcattggc ggaggcctgc ctttaccgcc gctcagatca tcaattacac catcagatgc
     4921 aaccctgtgg gcctgcagaa tgccagcctg gtcctgtatc tgcagaccag cgaaacccac
     4981 atgctggtgc agggactcga gcccaacacc aaatatgagt tcgccgtcag actgcatgtg
     5041 gatcagctgt ctagtccttg gagccccgtg gtgtaccact ctacattgcc tgaagctcca
     5101 gctggccctc ctgtgggagt gaaagtgacc ctgatcgagg acgatacagc cctggtgtcc
     5161 tggaagcctc ctgatggacc tgagacagtg gtcacccggt acacaatcct gtacgcctcc
     5221 agaaaggcct ggatcgctgg cgaatggcag gtcttgcata gagaaggcgc catcaccatg
     5281 gcactgctgg aaaatctggt ggccggcaac gtgtacatcg tgaagatcag cgccagcaac
     5341 gaagtcggcg agggcccttt tagcaatagc gtggaactgg ccgtgctgcc caaagaaacc
     5401 agcgagagca accagcggcc taagagactg gattctgccg acgccaaggt gtactccggc
     5461 tactaccacc tggatcagaa atccatgaca ggcgcgccgg gcggcgacaa gacccacacc
     5521 tgccccccct gcccagctcc agaactgctg ggagggcctt ccgtgttcct gttccccccc
     5581 aagcccaagg acaccctgat gatcagcagg acccccgagg tgacctgcgt ggtggtggcc
     5641 gtgtcccacg aggacccaga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac
     5701 aacgccaaga ccaagcccag agaggagcag tacaacagca cctacagggt ggtgtccgtg
     5761 ctgaccgtgc tgcaccagga ctggctgaac ggcaaagaat acaagtgcaa agtctccaac
     5821 aaggccctgg ctgccccaat cgaaaagaca atcagcaagg ccaagggcca gccacgggag
     5881 ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgtccctg
     5941 acctgtctgg tgaagggctt ctaccccagc gatatcgccg tggagtggga gagcaacggc
     6001 cagcccgaga acaactacaa gaccaccccc ccagtgctgg acagcgacgg cagcttcttc
     6061 ctgtacagca agctgaccgt ggacaagtcc aggtggcagc agggcaacgt gttcagctgc
     6121 agcgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgag cctgagcccc
     6181 ggcggcagcg gtcttaatga tattttcgag gctcagaaga ttgagtggca tgaacaccac
     6241 caccatcacc actaaggccg gcctgtagtc actggcacaa cgcgttaccc ggtaagccaa
     6301 tcgggtatac acggtcgtca tactgcagac agggttcttc tactttgcaa gatagtcttg
     6361 agtagtaaaa taaatagata gagaaaatca gcctcgactg tgccttctag ttgccagcca
     6421 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     6481 ctttcctaat aaaatgagga aattgcatca caacactcaa ccctatctcg gtctattctt
     6541 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     6601 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     6661 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     6721 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     6781 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     6841 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     6901 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     6961 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa
     7021 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatgacc
     7081 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     7141 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     7201 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     7261 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     7321 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     7381 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     7441 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     7501 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagaca
     7561 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     7621 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacacgtg
     7681 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     7741 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     7801 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     7861 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     7921 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatta
     7981 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     8041 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     8101 cgctgcgatg ataccgcgag aaccacgctc accggctccg gatttatcag caataaacca
     8161 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     8221 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     8281 tgttgccatc gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     8341 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     8401 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     8461 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     8521 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     8581 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     8641 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     8701 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     8761 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     8821 gaaatgttga atactcatat tcttcctt
//
