LOCUS       sequence-303125-        8944 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 8944)
  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 8944)
  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-SEMA5A-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8944
                     /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             6286..6330
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6331..6348
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6488..6505)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6494..6605
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6723..6743)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6728..7057
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6908..7043
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6970..6989
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7105..7152
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7171..7770
                     /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(7171..7190)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7667..7687
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7900..8021
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7937..7956)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7991..8010
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(8074..8865,8866..8934))
                     /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     8697..8716
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8935..8944,1..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 cggccgccga agctcaggga
     2761 acaacacagt gtcagagaac agagcacccc gtgatcagct acaaagagat cggcccctgg
     2821 ctgagagagt tcagagccaa gaacgccgtg gacttcagcc agctgacctt cgatcctggc
     2881 cagaaagagc tggtcgtcgg cgccagaaac tacctgtttc ggctgcagct ggaagatctg
     2941 agcctgatcc aggccgtgga atgggagtgt gatgaggcca ccaagaaggc ctgctacagc
     3001 aagggcaaga gcaaagagga atgccagaac tacatccggg tgctgctcgt tggcggcgac
     3061 agactgttta cctgcggcac caatgccttc acacccgtgt gcaccaacag aagcctgagc
     3121 aacctgaccg agatccacga ccagatcagc ggcatggcca gatgtcctta cagccctcag
     3181 cacaacagca cagctctgct gacagccggc ggagaactgt atgccgccac agccatggat
     3241 ttccctggca gagatcccgc catctacaga tctctgggaa tcctgcctcc tctgagaaca
     3301 gcccagtaca acagcaagtg gctgaacgag cccaacttcg tgtccagcta cgacatcggc
     3361 aacttcacct actttttctt ccgggaaaat gccgtggaac acgactgcgg caagaccgtg
     3421 tttagcagag ccgccagagt gtgcaagaac gatatcggcg gcagattcct gctcgaggac
     3481 acctggacaa cctttatgaa ggcccggctg aactgcagca gacctggcga ggtgcccttc
     3541 tactacaacg agctgcagag cacattcttt ctgcccgagc tggacctgat ctacggcatc
     3601 ttcaccacca acgtgaactc tatcgccgcc agcgccgtgt gcgtgttcaa tctgtctgct
     3661 atcgcccagg ccttcagcgg ccctttcaag taccaagaga acagcagaag cgcctggctg
     3721 ccctatccta atccaaatcc tcacttccag tgcggcaccg tggatcaggg cctgtatgtg
     3781 aacctgacag agagaaacct gcaggacgcc cagaaattca tcctgatgca cgaggtggtg
     3841 cagcccgtga ccacagtgcc tagcttcatg gaagataaca gccggttctc ccacgtggcc
     3901 gtggatgtgg ttcagggaag agaagccctg gtgcacatca tctacctggc caccgactac
     3961 ggcaccatca agaaagtgcg ggtgcccctg aatcagacca gcagctcttg tctgctggaa
     4021 gagatcgagc tgttccccga gcggagaaga gagcctatca gatccctgca gatcctgcac
     4081 agccagtccg tgctgtttgt gggcctgaga gaacacgtgg tcaagatccc tctgaagcgg
     4141 tgccagttct accggaccag aagcacctgt attggcgctc aggaccctta ctgtggctgg
     4201 gacgtcgtga tgaagaagtg caccagcctg gaagagtccc tgagcatgac acagtgggag
     4261 cagagcatca gcgcctgtcc taccagaaat ctgaccgtgg atggccactt tggcgtgtgg
     4321 tcaccttgga caccttgcac acacacagat ggctctgccg tgggcagctg tctgtgcaga
     4381 acaagaagct gcgatagccc tgctcctcag tgtggcggat ggcaatgtga aggccctggc
     4441 atggaaatcg ccaactgtag cagaaatggc ggctggaccc catggacctc ttggagccct
     4501 tgtagcacaa cctgtggcat cggctttcaa gtgcggcaga gaagctgcag caaccccaca
     4561 cctagacacg gcggaagagt gtgcgtgggc cagaacagag aggaacggta ctgcaacgag
     4621 catctgctgt gccctccaca catgttttgg acaggatggg gcccctggga gagatgtaca
     4681 gcacaatgtg gcggaggaat ccaggccaga agaaggatct gcgagaacgg ccctgattgt
     4741 gccggctgca atgtggaata ccagagctgc aacacaaacc cctgtcctga gctgaagaaa
     4801 acaacaccct ggactccctg gacacccgtg aacatctctg acaatggcgg ccactacgag
     4861 cagcggttca gatacacctg taaagcccgg ctggccgatc ctaacctgct tgaagtgggc
     4921 agacagcgga tcgagatgag atactgtagc tccgatggca ccagcggctg tagcacagat
     4981 ggactgagcg gcgattttct gagagccggc agatacagcg cccacacagt taatggtgct
     5041 tggagcgcct ggacaagctg gtcccaatgt agcagggatt gcagcagagg catccggaac
     5101 cggaagagag tgtgtaacaa ccccgagcct aagtacggcg gcatgccttg tcttggccct
     5161 agcctggaat atcaagagtg caatatcctg ccttgtcctg tggacggcgt ctggtcttgt
     5221 tggagtcctt ggaccaagtg ctctgccaca tgcggcggag gccactacat gagaaccaga
     5281 tcctgtagca accccgctcc tgcctacggc ggagatattt gtctgggact gcataccgaa
     5341 gaggccctgt gcaataccca gccatgtcca gaatcttgga gcgagtggtc cgattggagc
     5401 gaatgtgaag ccagcggcgt gcaagttcgg gccagacagt gtatcctgct gttccctatg
     5461 ggctcccagt gcagcggcaa taccaccgaa agcagaccct gcgtgttcga cagcaacttc
     5521 atccctgagg tgtccgtggc cagatccagc agcgtggaag agaaaagatg cggcgagttc
     5581 aacatgggcg cgccgggcgg cgacaagacc cacacctgcc ccccctgccc agctccagaa
     5641 ctgctgggag ggccttccgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc
     5701 agcaggaccc ccgaggtgac ctgcgtggtg gtggccgtgt cccacgagga cccagaggtg
     5761 aagttcaact ggtacgtgga cggcgtggag gtgcacaacg ccaagaccaa gcccagagag
     5821 gagcagtaca acagcaccta cagggtggtg tccgtgctga ccgtgctgca ccaggactgg
     5881 ctgaacggca aagaatacaa gtgcaaagtc tccaacaagg ccctggctgc cccaatcgaa
     5941 aagacaatca gcaaggccaa gggccagcca cgggagcccc aggtgtacac cctgcccccc
     6001 agccgggagg agatgaccaa gaaccaggtg tccctgacct gtctggtgaa gggcttctac
     6061 cccagcgata tcgccgtgga gtgggagagc aacggccagc ccgagaacaa ctacaagacc
     6121 acccccccag tgctggacag cgacggcagc ttcttcctgt acagcaagct gaccgtggac
     6181 aagtccaggt ggcagcaggg caacgtgttc agctgcagcg tgatgcacga ggccctgcac
     6241 aaccactaca cccagaagtc cctgagcctg agccccggcg gcagcggtct taatgatatt
     6301 ttcgaggctc agaagattga gtggcatgaa caccaccacc atcaccacta aggccggcct
     6361 gtagtcactg gcacaacgcg ttacccggta agccaatcgg gtatacacgg tcgtcatact
     6421 gcagacaggg ttcttctact ttgcaagata gtcttgagta gtaaaataaa tagatagaga
     6481 aaatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     6541 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     6601 gcatcacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     6661 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     6721 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     6781 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     6841 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     6901 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     6961 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     7021 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     7081 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata
     7141 gtataatacg acaaggtgag gaactaaacc atgaccgagt acaagcccac ggtgcgcctc
     7201 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     7261 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     7321 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     7381 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     7441 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     7501 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     7561 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     7621 gccgagcgcg ccggggtgcc cgccttcctg gagacatccg cgccccgcaa cctccccttc
     7681 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     7741 tggtgcatga cccgcaagcc cggtgcctga cacgtgctac gagatttcga ttccaccgcc
     7801 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     7861 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     7921 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     7981 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     8041 acctctagct agagcttggc gtaatcatgg tcattaccaa tgcttaatca gtgaggcacc
     8101 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     8161 aactacgata cgggagggct taccatctgg ccccagcgct gcgatgatac cgcgagaacc
     8221 acgctcaccg gctccggatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     8281 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     8341 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccatcgcta caggcatcgt
     8401 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     8461 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     8521 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     8581 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     8641 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     8701 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     8761 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     8821 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     8881 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatattctt
     8941 cctt
//
