LOCUS       sequence-431377-        6997 bp    DNA     circular SYN 14-APR-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 6997)
  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 6997)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-KIR3DL3.2-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     815..832
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             2573..2617
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2618..2635
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2775..2792)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2781..2892
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3010..3030)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3015..3344
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3195..3330
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3257..3276
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3392..3439
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3458..4057
                     /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(3458..3477)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3954..3974
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4187..4308
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4224..4243)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4278..4297
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(4361..5152,5153..5221))
                     /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     4984..5003
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5222..5313)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5355..5374
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5380..5399
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5485..6073
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5974..5993
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6227..6244
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6342..6359
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6343..6359
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6815..6831
                     /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)."
ORIGIN
        1 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
      901 cgaaaacgcg cctgctagcg ccaccatgaa gaccatcatt gccctgagct acatcttctg
      961 cctggtgttc gctgcggccg cccaagataa gccttttctg tctgcttggc ctggcaccgt
     1021 ggtgtctgaa ggacagcatg tgaccctgca gtgcagaagc agactgggct tcaacgagtt
     1081 cagcctgagc aaagaggacg gcatgcctgt gcctgagctg tacaaccgga tcttccggaa
     1141 cagcttcctg atgggccctg tgacaccagc tcacgccggc acatacagat gctgtagctc
     1201 tcaccctcac agccccacag gatggtccgc tccatctaac cctgtggtca tcatggtcac
     1261 cggcgtgcac agaaagcctt ctctgcttgc ccatcctgga cctctggtca agtctggcga
     1321 gacagtgatt ctgcagtgtt ggagcgacgt ccgcttcgag agattcctgc tgcacagaga
     1381 gggcatcaca gaggaccctc tgagactcgt gggacagctg catgatgccg gcagccaagt
     1441 gaattacagc atgggcccaa tgacacccgc actggccggc acctatagat gtttcggcag
     1501 cgtgacccat ctgccttacg agctgtctgc ccctagcgac cctctggata ttgtggttgt
     1561 gggcctgtac ggcaagccta gcctttctgc tcagcctgga cctacagtgc aggccggcga
     1621 aaatgtgaca ctgagctgtt ccagcagaag cctgttcgac atctaccacc tgtccagaga
     1681 agccgaagcc ggcgaactga gactgacagc cgtgctgaga gtgaacggca ccttccaggc
     1741 caattttcca ctgggaccag tgacacacgg cggcaattac cggtgcttcg gctcttttag
     1801 agccctgcct catgcttgga gcgatccctc tgatcctctg cctgtgtctg tgaccggcaa
     1861 cagcagacac cttggcgcgc cgggcggcga caagacccac acctgccccc cctgcccagc
     1921 tccagaactg ctgggagggc cttccgtgtt cctgttcccc cccaagccca aggacaccct
     1981 gatgatcagc aggacccccg aggtgacctg cgtggtggtg gccgtgtccc acgaggaccc
     2041 agaggtgaag ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc
     2101 cagagaggag cagtacaaca gcacctacag ggtggtgtcc gtgctgaccg tgctgcacca
     2161 ggactggctg aacggcaaag aatacaagtg caaagtctcc aacaaggccc tggctgcccc
     2221 aatcgaaaag acaatcagca aggccaaggg ccagccacgg gagccccagg tgtacaccct
     2281 gccccccagc cgggaggaga tgaccaagaa ccaggtgtcc ctgacctgtc tggtgaaggg
     2341 cttctacccc agcgatatcg ccgtggagtg ggagagcaac ggccagcccg agaacaacta
     2401 caagaccacc cccccagtgc tggacagcga cggcagcttc ttcctgtaca gcaagctgac
     2461 cgtggacaag tccaggtggc agcagggcaa cgtgttcagc tgcagcgtga tgcacgaggc
     2521 cctgcacaac cactacaccc agaagtccct gagcctgagc cccggcggca gcggtcttaa
     2581 tgatattttc gaggctcaga agattgagtg gcatgaacac caccaccatc accactaagg
     2641 ccggcctgta gtcactggca caacgcgtta cccggtaagc caatcgggta tacacggtcg
     2701 tcatactgca gacagggttc ttctactttg caagatagtc ttgagtagta aaataaatag
     2761 atagagaaaa tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     2821 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     2881 ggaaattgca tcacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2941 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3001 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     3061 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     3121 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     3181 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3241 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     3301 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     3361 gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg catagtatat
     3421 cggcatagta taatacgaca aggtgaggaa ctaaaccatg accgagtaca agcccacggt
     3481 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
     3541 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
     3601 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     3661 cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     3721 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     3781 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     3841 cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     3901 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acatccgcgc cccgcaacct
     3961 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     4021 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacac gtgctacgag atttcgattc
     4081 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4141 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4201 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4261 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4321 accgtcgacc tctagctaga gcttggcgta atcatggtca ttaccaatgc ttaatcagtg
     4381 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4441 tgtagataac tacgatacgg gagggcttac catctggccc cagcgctgcg atgataccgc
     4501 gagaaccacg ctcaccggct ccggatttat cagcaataaa ccagccagcc ggaagggccg
     4561 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4621 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc atcgctacag
     4681 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4741 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4801 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4861 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4921 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4981 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5041 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5101 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5161 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5221 tattcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5281 acatatttga atgtatttag aaaaataaac aaataggggt cagtgttaca accaattaac
     5341 caattctgaa cattatcgcg agcccattta tacctgaata tggctcataa caccccttgc
     5401 tcatgaccaa aatcccttaa cgtgagttac gcgcgcgtcg ttccactgag cgtcagaccc
     5461 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5521 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5581 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     5641 gtagccgtag ttagcccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5701 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5761 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5821 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5881 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5941 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6001 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6061 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6121 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     6181 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     6241 cgaggaagcg gaaggcgaga gtagggaact gccaggcatc aaactaagca gaaggcccct
     6301 gacggatggc ctttttgcgt ttctacaaac tctttctgtg ttgtaaaacg acggccagtc
     6361 ttaagctcgg gccccctggg cggttctgat aacgagtaat cgttaatccg caaataacgt
     6421 aaaaacccgc ttcggcgggt ttttttatgg ggggagttta gggaaagagc atttgtcaga
     6481 atatttaagg gcgcctgtca ctttgcttga tatatgagaa ttatttaacc ttataaatga
     6541 gaaaaaagca acgcacttta aataagatac gttgcttttt cgattgatga acacctataa
     6601 ttaaactatt catctattat ttatgatttt ttgtatatac aatatttcta gtttgttaaa
     6661 gagaattaag aaaataaatc tcgaaaataa taaagggaaa atcagttttt gatatcaaaa
     6721 ttatacatgt caacgataat acaaaatata atacaaacta taagatgtta tcagtattta
     6781 ttatcattta gaataaattt tgtgtcgccc ttaattgtga gcggataaca attacgagct
     6841 tcatgcacag tggcgttgac tcaatattgg ccattagcca tattattcat tggttatata
     6901 gcataaatca atattggcta ttggccattg catacgttgt atctatatca taatatgtac
     6961 atttatattg gctcatgtcc aatatgaccg ccatgtt
//
