LOCUS       sequence-302529-        6913 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 6913)
  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 6913)
  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-SPACA6-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6913
                     /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             4255..4299
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4300..4317
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4457..4474)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4463..4574
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4692..4712)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4697..5026
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4877..5012
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4939..4958
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5074..5121
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5140..5739
                     /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(5140..5159)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5636..5656
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5869..5990
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5906..5925)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5960..5979
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6043..6834,6835..6903))
                     /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     6666..6685
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6904..6913,1..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 cggccgcctg tctgctgtgt
     2761 tttaccacct acagcgagcg gctgcggatc tgccagatgt ttgtgggcat gagaagcccc
     2821 aagctggaag agtgcgagga agcctttaca gccgcctttc agggcctgag cgacaccgag
     2881 atcaactacg acgagagaag ccatctgcac gacaccttca cacagatgac acacgccctg
     2941 caagaactgg ccgctgctca gggaagcttt gaggtggcat ttcctgacgc cgccgagaag
     3001 atgaagaaag tgatcaccca gctgaaagag gcccaggcct gcattcctcc atgcggcctt
     3061 caagagttcg ccagacggtt tctgtgcagc ggctgctaca gcagagtgtg cgatctgcct
     3121 ctggactgcc ctgtgcagga tgtgacagtg acaagaggcg accaggccat gttcagctgc
     3181 atcgtgaact tccagctgcc taaagaggaa atcacctact cttggaagtt tgccggcgga
     3241 ggcctgagaa cccaggacct gagctacttc agagacatgc ccagagccga gggctacctg
     3301 gccagaatta gacctgctca gctgacccac agaggcacct tcagttgcgt gatcaagcag
     3361 gatcagcggc ccctggctag actgtacttc ttcctgaatg tgaccggacc tcctccaaga
     3421 gccgaaacag aactgcaggc cagcttccgg gaagtgctta gatgggcccc tagagatgcc
     3481 gagctgattg agccttggag gccttctctg ggagagctgc ttgctagacc tgaggctctg
     3541 acccctagca accttggcgc gccgggcggc gacaagaccc acacctgccc cccctgccca
     3601 gctccagaac tgctgggagg gccttccgtg ttcctgttcc cccccaagcc caaggacacc
     3661 ctgatgatca gcaggacccc cgaggtgacc tgcgtggtgg tggccgtgtc ccacgaggac
     3721 ccagaggtga agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag
     3781 cccagagagg agcagtacaa cagcacctac agggtggtgt ccgtgctgac cgtgctgcac
     3841 caggactggc tgaacggcaa agaatacaag tgcaaagtct ccaacaaggc cctggctgcc
     3901 ccaatcgaaa agacaatcag caaggccaag ggccagccac gggagcccca ggtgtacacc
     3961 ctgcccccca gccgggagga gatgaccaag aaccaggtgt ccctgacctg tctggtgaag
     4021 ggcttctacc ccagcgatat cgccgtggag tgggagagca acggccagcc cgagaacaac
     4081 tacaagacca cccccccagt gctggacagc gacggcagct tcttcctgta cagcaagctg
     4141 accgtggaca agtccaggtg gcagcagggc aacgtgttca gctgcagcgt gatgcacgag
     4201 gccctgcaca accactacac ccagaagtcc ctgagcctga gccccggcgg cagcggtctt
     4261 aatgatattt tcgaggctca gaagattgag tggcatgaac accaccacca tcaccactaa
     4321 ggccggcctg tagtcactgg cacaacgcgt tacccggtaa gccaatcggg tatacacggt
     4381 cgtcatactg cagacagggt tcttctactt tgcaagatag tcttgagtag taaaataaat
     4441 agatagagaa aatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     4501 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     4561 gaggaaattg catcacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     4621 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4681 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     4741 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     4801 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     4861 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     4921 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     4981 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     5041 ttgtatatcc attttcggat ctgatcagca cgtgttgaca attaatcatc ggcatagtat
     5101 atcggcatag tataatacga caaggtgagg aactaaacca tgaccgagta caagcccacg
     5161 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     5221 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     5281 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     5341 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     5401 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     5461 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     5521 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     5581 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacatccgc gccccgcaac
     5641 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     5701 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac acgtgctacg agatttcgat
     5761 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     5821 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     5881 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     5941 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     6001 ataccgtcga cctctagcta gagcttggcg taatcatggt cattaccaat gcttaatcag
     6061 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6121 cgtgtagata actacgatac gggagggctt accatctggc cccagcgctg cgatgatacc
     6181 gcgagaacca cgctcaccgg ctccggattt atcagcaata aaccagccag ccggaagggc
     6241 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6301 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccatcgctac
     6361 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6421 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6481 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6541 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6601 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6661 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6721 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6781 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6841 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6901 catattcttc ctt
//
