LOCUS       sequence-302321-        6691 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 6691)
  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 6691)
  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-RAET1E-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6691
                     /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             4033..4077
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4078..4095
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4235..4252)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4241..4352
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4470..4490)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4475..4804
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4655..4790
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4717..4736
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4852..4899
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4918..5517
                     /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(4918..4937)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5414..5434
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5647..5768
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5684..5703)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5738..5757
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5821..6612,6613..6681))
                     /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     6444..6463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6682..6691,1..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 cggccgccca tagcctgtgc
     2761 ttcaacttca ccatcaagag cctgagcaga cccggacagc cttggtgcga agcccaggtg
     2821 ttcctgaaca agaacctgtt cctgcagtac aacagcgaca acaacatggt caagcctctg
     2881 ggcctgctgg gcaagaaggt gtacgctaca tctacctggg gcgagctgac acagacactg
     2941 ggcgaagtgg gcagagatct gagaatgctg ctgtgcgaca tcaagcccca gatcaagacc
     3001 agcgatccca gcacactgca ggtcgagatg ttctgtcagc gcgaggccga aagatgtaca
     3061 ggcgcctctt ggcagttcgc caccaatggc gagaagtccc tgctgttcga cgccatgaat
     3121 atgacctgga ccgtgatcaa ccacgaggcc agcaagatca aagagacatg gaagaaggac
     3181 cgcggcctgg aaaagtactt cagaaagctg agcaagggcg actgcgacca ctggctgaga
     3241 gaatttctcg gccactggga agccatgcct gagcctacag tgtctcccgt gaacgccagc
     3301 gatatccact ggtctagcag cagcctgcct gatggcgcgc cgggcggcga caagacccac
     3361 acctgccccc cctgcccagc tccagaactg ctgggagggc cttccgtgtt cctgttcccc
     3421 cccaagccca aggacaccct gatgatcagc aggacccccg aggtgacctg cgtggtggtg
     3481 gccgtgtccc acgaggaccc agaggtgaag ttcaactggt acgtggacgg cgtggaggtg
     3541 cacaacgcca agaccaagcc cagagaggag cagtacaaca gcacctacag ggtggtgtcc
     3601 gtgctgaccg tgctgcacca ggactggctg aacggcaaag aatacaagtg caaagtctcc
     3661 aacaaggccc tggctgcccc aatcgaaaag acaatcagca aggccaaggg ccagccacgg
     3721 gagccccagg tgtacaccct gccccccagc cgggaggaga tgaccaagaa ccaggtgtcc
     3781 ctgacctgtc tggtgaaggg cttctacccc agcgatatcg ccgtggagtg ggagagcaac
     3841 ggccagcccg agaacaacta caagaccacc cccccagtgc tggacagcga cggcagcttc
     3901 ttcctgtaca gcaagctgac cgtggacaag tccaggtggc agcagggcaa cgtgttcagc
     3961 tgcagcgtga tgcacgaggc cctgcacaac cactacaccc agaagtccct gagcctgagc
     4021 cccggcggca gcggtcttaa tgatattttc gaggctcaga agattgagtg gcatgaacac
     4081 caccaccatc accactaagg ccggcctgta gtcactggca caacgcgtta cccggtaagc
     4141 caatcgggta tacacggtcg tcatactgca gacagggttc ttctactttg caagatagtc
     4201 ttgagtagta aaataaatag atagagaaaa tcagcctcga ctgtgccttc tagttgccag
     4261 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4321 gtcctttcct aataaaatga ggaaattgca tcacaacact caaccctatc tcggtctatt
     4381 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4441 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     4501 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     4561 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4621 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4681 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4741 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4801 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat
     4861 taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg
     4921 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     4981 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     5041 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     5101 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     5161 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     5221 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     5281 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     5341 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     5401 acatccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     5461 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacac
     5521 gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     5581 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     5641 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     5701 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5761 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     5821 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5881 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5941 cagcgctgcg atgataccgc gagaaccacg ctcaccggct ccggatttat cagcaataaa
     6001 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6061 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6121 cgttgttgcc atcgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6181 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6241 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6301 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6361 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6421 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6481 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6541 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6601 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6661 acggaaatgt tgaatactca tattcttcct t
//
