LOCUS       sequence-304343-        8542 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 8542)
  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 8542)
  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-mLepr-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8542
                     /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     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             5884..5928
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5929..5946
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6086..6103)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6092..6203
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6321..6341)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6326..6655
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6506..6641
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6568..6587
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6703..6750
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6769..7368
                     /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(6769..6788)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7265..7285
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7498..7619
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7535..7554)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7589..7608
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7672..8463,8464..8532))
                     /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     8295..8314
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8533..8542,1..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 cggccgccta tccgatctcc
     2761 ccatggaaat ttaagttgtt ttgtggaccg ccgaatacca ccgatgactc ttttctttca
     2821 ccagcggggg cacctaataa tgcatcagcg ctgaaggggg cgtctgaagc tatagtcgag
     2881 gccaaattca acagttctgg gatttatgtg ccagagcttt ccaaaactgt cttccactgc
     2941 tgtttcggca atgaacaagg ccagaattgc tctgcactga ctgataatac agaagggaaa
     3001 acactcgcat cagtggtgaa ggccagcgta ttcaggcaac tgggggttaa ttgggacatc
     3061 gagtgctgga tgaaaggtga tctcactctg ttcatatgcc acatggagcc attgcctaaa
     3121 aatccgttca aaaactacga cagtaaagta catttgctgt atgacctgcc tgaggtgatc
     3181 gacgattctc cgttgcctcc gctgaaggac agcttccaaa ctgtccagtg taattgctcc
     3241 ttgcgagggt gtgagtgtca tgtacccgta cccagggcta agctcaatta cgctctcttg
     3301 atgtacttgg aaataactag tgctggcgta tccttccaat caccactcat gtcattgcaa
     3361 ccgatgctgg tcgtgaaacc ggaccctcca cttggattgc atatggaagt gacggacgat
     3421 gggaacctta agatttcttg ggattctcag acgatggcgc cttttcccct gcagtaccag
     3481 gtaaagtact tggagaatag tacaattgta agggaggcag cggagatagt cagtgcgact
     3541 tccctgcttg tcgatagcgt cctgccgggc tcatcatatg aagtacaggt aaggagcaaa
     3601 cggctggacg ggagtggggt ctggtccgat tggagctctc cacaagtttt tacgacgcaa
     3661 gacgtagtct atttccctcc aaaaatcctt acgagcgtag gcagcaacgc ctccttccat
     3721 tgtatttata aaaacgagaa ccaaatcatc tcaagcaagc agatcgtttg gtggcggaac
     3781 ctggcggaaa agatcccaga gattcaatat tcaatcgtca gtgaccgagt gtccaaggta
     3841 accttttcca atctgaaagc aacgcgacct aggggaaaat tcacgtatga tgccgtatat
     3901 tgctgtaacg aacaagcttg tcatcatcgg tacgcagagt tgtacgtgat tgatgtcaac
     3961 atcaacatat cctgcgagac agacggttac ctgaccaaga tgacttgtcg ctggtcccca
     4021 tccacgatcc agagtcttgt gggcagtacg gtccaacttc gctaccatcg acgaagcctc
     4081 tactgcccag actctcctag catacaccca acatctgaac caaaaaactg cgtcctccaa
     4141 agagacgggt tctatgagtg cgttttccaa ccaatatttc ttttgtccgg ctatacgatg
     4201 tggatcagaa tcaaccattc cctgggttcc ctcgattctc cacccacgtg tgttttgccc
     4261 gactctgttg tcaaacctct tccaccctca aatgtaaagg cggaaataac cgtcaatacg
     4321 ggtcttctta aagtcagctg ggagaaacca gtttttccgg aaaacaactt gcaattccaa
     4381 atacggtacg gactctctgg caaagaaatc caatggaaga ctcacgaggt ttttgacgcg
     4441 aagagtaaat ccgcgtccct tttggtatct gacctctgcg cagtatacgt tgttcaggtt
     4501 cgatgtagaa ggcttgatgg actgggatat tggtctaatt ggtcaagccc agcgtacacg
     4561 ttggtcatgg acgtaaaggt gcctatgcga gggcccgagt tctggcggaa aatggacgga
     4621 gatgttacaa agaaagaacg aaatgtaacg cttctttgga aacccctcac taaaaacgat
     4681 tccctgtgct cagtacggag atacgtcgtg aagcatcgca cggcccataa cggtacatgg
     4741 tctgaagatg taggtaatcg aacgaacttg acgtttctgt ggacggaacc tgcacacaca
     4801 gttactgtct tggcagtaaa ctccctcggc gccagtttgg tcaactttaa cttgacgttc
     4861 tcttggccca tgtctaaagt aagcgcggtg gaatcattga gcgcttatcc tctgtccagt
     4921 tcatgtgtca tcctcagttg gacgctcagc ccagatgatt acagtctcct ctatctggtg
     4981 atcgagtgga agattttgaa cgaagatgac gggatgaagt ggctccgaat accctccaac
     5041 gtgaagaaat tttatataca tgataacttc attccaatag agaaatatca gttctctctt
     5101 tatccggtat ttatggaggg ggtgggtaaa cctaagatca taaatggctt cacaaaagat
     5161 gcgattgaca aacaacaaaa cgatggcgcg ccgggcggcg acaagaccca cacctgcccc
     5221 ccctgcccag ctccagaact gctgggaggg ccttccgtgt tcctgttccc ccccaagccc
     5281 aaggacaccc tgatgatcag caggaccccc gaggtgacct gcgtggtggt ggccgtgtcc
     5341 cacgaggacc cagaggtgaa gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc
     5401 aagaccaagc ccagagagga gcagtacaac agcacctaca gggtggtgtc cgtgctgacc
     5461 gtgctgcacc aggactggct gaacggcaaa gaatacaagt gcaaagtctc caacaaggcc
     5521 ctggctgccc caatcgaaaa gacaatcagc aaggccaagg gccagccacg ggagccccag
     5581 gtgtacaccc tgccccccag ccgggaggag atgaccaaga accaggtgtc cctgacctgt
     5641 ctggtgaagg gcttctaccc cagcgatatc gccgtggagt gggagagcaa cggccagccc
     5701 gagaacaact acaagaccac ccccccagtg ctggacagcg acggcagctt cttcctgtac
     5761 agcaagctga ccgtggacaa gtccaggtgg cagcagggca acgtgttcag ctgcagcgtg
     5821 atgcacgagg ccctgcacaa ccactacacc cagaagtccc tgagcctgag ccccggcggc
     5881 agcggtctta atgatatttt cgaggctcag aagattgagt ggcatgaaca ccaccaccat
     5941 caccactaag gccggcctgt agtcactggc acaacgcgtt acccggtaag ccaatcgggt
     6001 atacacggtc gtcatactgc agacagggtt cttctacttt gcaagatagt cttgagtagt
     6061 aaaataaata gatagagaaa atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     6121 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     6181 taataaaatg aggaaattgc atcacaacac tcaaccctat ctcggtctat tcttttgatt
     6241 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     6301 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     6361 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     6421 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     6481 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     6541 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     6601 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     6661 cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg
     6721 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat gaccgagtac
     6781 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     6841 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     6901 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     6961 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     7021 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     7081 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     7141 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     7201 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacatccgcg
     7261 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     7321 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaca cgtgctacga
     7381 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     7441 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     7501 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     7561 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     7621 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc attaccaatg
     7681 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     7741 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagcgctgc
     7801 gatgataccg cgagaaccac gctcaccggc tccggattta tcagcaataa accagccagc
     7861 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     7921 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     7981 catcgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     8041 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     8101 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     8161 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     8221 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     8281 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     8341 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     8401 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     8461 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     8521 ttgaatactc atattcttcc tt
//
