LOCUS       sequence-302961-        7924 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 7924)
  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 7924)
  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-DNER-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..7924
                     /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"
     regulatory      4419..4428
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5266..5310
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5311..5328
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5468..5485)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5474..5585
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5703..5723)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5708..6037
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5888..6023
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5950..5969
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6085..6132
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6151..6750
                     /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(6151..6170)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6647..6667
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6880..7001
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6917..6936)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6971..6990
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7054..7845,7846..7914))
                     /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     7677..7696
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7915..7924,1..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 cggccgccaa tcctgttcct
     2761 gctgctcctc tttctgcccc tggaccttgt gctgcccagc cttgtagaaa tggcggcgtg
     2821 tgtaccagca gacccgagcc tgatcctcag catcctgctc cagctggcga gcctggctac
     2881 tcttgtacat gtcctgccgg aatcagcggc gccaattgtc agctggtggc cgatccttgt
     2941 gccagcaatc cttgccacca cggcaattgc agcagcagct ctagcagctc ctccgacggc
     3001 tacctgtgca tctgcaatga gggctacgag ggccccaatt gcgaacaggc tctgccttct
     3061 ctgcctgcca ctggctggac agaatctatg gcccctagac agctgcagcc cgtgccagct
     3121 acacaagagc ccgataagat cctgcctcgg agccaggcta cagtgaccct gcctacatgg
     3181 cagcctaaga ccggccagaa agtggtggaa atgaagtggg accaagtgga agtgatcccc
     3241 gatatcgcct gcggcaacgc cagcagcaat tcttctgctg gcggcagact ggtgtccttc
     3301 gaggtgccac agaacaccag cgtgaagatc agacaggatg ccaccgccag cctgattctg
     3361 ctgtggaaag tgaccgccac cggcttccag cagtgctctc tgatcgatgg cagaagcgtg
     3421 acacccctgc aagcaagcgg aggactggtg ctgctcgaag aaatgctggc cctgggcaac
     3481 aaccacttca tcggcttcgt gaacgacagc gtgaccaaga gcatcgtggc cctgagactg
     3541 accctggtgg tcaaggtgtc cacatgtgtg cctggcgaga gccacgccaa cgatctggaa
     3601 tgttctggca agggcaagtg caccaccaag cctagcgaag ccaccttcag ctgtacctgc
     3661 gaggaacagt acgtgggcac cttctgcgaa gagtacgacg cctgccagag aaagccctgt
     3721 cagaacaacg cctcctgcat cgacgccaat gagaagcagg acggcagcaa cttcacctgt
     3781 gtgtgtctgc ctggctatac cggcgagctg tgccagagca agatcgacta ctgcattctg
     3841 gacccctgca gaaacggcgc cacctgtatc tctagcctga gcggctttac ctgtcagtgc
     3901 cccgagggct attttggcag cgcctgtgaa gagaaggtgg acccttgtgc aagctcccca
     3961 tgccaaaaca acggcacctg ttacgtggac ggcgtgcact ttacctgcaa ctgcagccca
     4021 ggcttcactg gccctacatg tgcccagctg atcgacttct gtgctctgag cccttgcgct
     4081 cacggcacat gcagatctgt gggcaccagc tacaagtgcc tgtgcgatcc tggctatcac
     4141 ggcctgtatt gcgaagagga gtacaacgag tgcctgagcg ccccttgtct gaatgccgcc
     4201 acttgcagag atctggtcaa cggctacgag tgcgtgtgtc tggccgagta caagggcacc
     4261 cactgcgagc tgtacaagga cccatgtgcc aacgtgtcct gcctgaacgg cgctacatgt
     4321 gatagcgacg gcctgaatgg aacctgcatc tgcgcccctg ggtttaccgg cgaagagtgc
     4381 gatatcgaca tcaatgagtg cgacagcaac ccctgtcacc atggcggctc ttgtctggac
     4441 cagcctaacg gctacaactg ccactgtcct cacggatggg tcggagccaa ctgcgagatt
     4501 catctgcagt ggaagtccgg ccacatggcc gagagcctga ccaacatgcc tcggcacagt
     4561 ctgtatggcg cgccgggcgg cgacaagacc cacacctgcc ccccctgccc agctccagaa
     4621 ctgctgggag ggccttccgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc
     4681 agcaggaccc ccgaggtgac ctgcgtggtg gtggccgtgt cccacgagga cccagaggtg
     4741 aagttcaact ggtacgtgga cggcgtggag gtgcacaacg ccaagaccaa gcccagagag
     4801 gagcagtaca acagcaccta cagggtggtg tccgtgctga ccgtgctgca ccaggactgg
     4861 ctgaacggca aagaatacaa gtgcaaagtc tccaacaagg ccctggctgc cccaatcgaa
     4921 aagacaatca gcaaggccaa gggccagcca cgggagcccc aggtgtacac cctgcccccc
     4981 agccgggagg agatgaccaa gaaccaggtg tccctgacct gtctggtgaa gggcttctac
     5041 cccagcgata tcgccgtgga gtgggagagc aacggccagc ccgagaacaa ctacaagacc
     5101 acccccccag tgctggacag cgacggcagc ttcttcctgt acagcaagct gaccgtggac
     5161 aagtccaggt ggcagcaggg caacgtgttc agctgcagcg tgatgcacga ggccctgcac
     5221 aaccactaca cccagaagtc cctgagcctg agccccggcg gcagcggtct taatgatatt
     5281 ttcgaggctc agaagattga gtggcatgaa caccaccacc atcaccacta aggccggcct
     5341 gtagtcactg gcacaacgcg ttacccggta agccaatcgg gtatacacgg tcgtcatact
     5401 gcagacaggg ttcttctact ttgcaagata gtcttgagta gtaaaataaa tagatagaga
     5461 aaatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     5521 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     5581 gcatcacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     5641 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     5701 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     5761 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     5821 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     5881 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     5941 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     6001 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     6061 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata
     6121 gtataatacg acaaggtgag gaactaaacc atgaccgagt acaagcccac ggtgcgcctc
     6181 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     6241 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     6301 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     6361 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     6421 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     6481 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     6541 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     6601 gccgagcgcg ccggggtgcc cgccttcctg gagacatccg cgccccgcaa cctccccttc
     6661 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     6721 tggtgcatga cccgcaagcc cggtgcctga cacgtgctac gagatttcga ttccaccgcc
     6781 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     6841 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     6901 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     6961 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     7021 acctctagct agagcttggc gtaatcatgg tcattaccaa tgcttaatca gtgaggcacc
     7081 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7141 aactacgata cgggagggct taccatctgg ccccagcgct gcgatgatac cgcgagaacc
     7201 acgctcaccg gctccggatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7261 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     7321 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccatcgcta caggcatcgt
     7381 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7441 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7501 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7561 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7621 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7681 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7741 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7801 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7861 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatattctt
     7921 cctt
//
