LOCUS       sequence-302879-        7603 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 7603)
  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 7603)
  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-KIT-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..7603
                     /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             4945..4989
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4990..5007
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5147..5164)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5153..5264
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5382..5402)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5387..5716
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5567..5702
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5629..5648
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5764..5811
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5830..6429
                     /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(5830..5849)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6326..6346
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6559..6680
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6596..6615)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6650..6669
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6733..7524,7525..7593))
                     /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     7356..7375
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7594..7603,1..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 accttctgtt
     2761 tctcctggcg aaccttctcc acctagcatt caccccggca agagcgacct gatcgttaga
     2821 gtgggcgacg agatccggct gctgtgtacc gatcctggct tcgtgaagtg gaccttcgag
     2881 atcctggacg agacaaacga gaacaagcag aacgagtgga tcaccgagaa ggccgaggcc
     2941 accaacaccg gcaagtacac ctgtaccaac aagcacggcc tgagcaacag catctacgtg
     3001 ttcgtgcggg accccgccaa gctgttcctg gtggatagaa gcctgtacgg caaagaggac
     3061 aacgacaccc tcgtgcggtg ccctctgaca gatcccgaag tgaccaacta cagcctgaag
     3121 ggctgtcagg gcaagcccct gcctaaggac ctgagattca tccccgatcc taaggccggc
     3181 atcatgatca agagcgtgaa gcgggcctac cacagactgt gcctgcactg ttccgtggac
     3241 caagagggca agtctgtgct gagcgagaag ttcatcctga aagtgcggcc tgccttcaag
     3301 gccgtgcctg tggtgtctgt gtctaaggcc agctacctgc tgagagaggg cgaagagttc
     3361 accgtgacct gcaccatcaa ggacgtgtcc agcagcgtgt acagcacctg gaagagagag
     3421 aacagccaga ccaagctgca agagaagtac aacagctggc accacggcga cttcaactac
     3481 gagagacagg ccacactgac catcagcagc gccagagtga atgacagcgg cgtgttcatg
     3541 tgctacgcca acaacacctt cggcagcgcc aacgtgacca ccacactgga agtggtggac
     3601 aagggcttca tcaacatctt ccccatgatc aacaccaccg tgtttgtgaa cgacggcgag
     3661 aacgtggacc tgattgtgga atacgaggcc tttcctaagc cagagcacca gcagtggatc
     3721 tacatgaacc ggaccttcac cgacaagtgg gaagattacc ccaagagcga gaacgagagc
     3781 aacatcagat acgtgtccga gctgcacctg accagactga agggaacaga aggcggcacc
     3841 tacacctttc tggtgtccaa ctccgacgtg aacgccgcca ttgccttcaa cgtgtacgtg
     3901 aacaccaagc ctgagatcct gacctacgac cggctggtca atggcatgct gcagtgtgtg
     3961 gccgctggct ttcctgagcc tacaatcgac tggtacttct gccctggcac cgagcagaga
     4021 tgtagcgctt ctgtgctgcc cgtggatgtg cagaccctga attctagcgg ccctccattc
     4081 ggcaagctgg tggtgcagag cagcatcgat agcagcgcct tcaagcacaa cggcaccgtg
     4141 gaatgcaagg cctacaacga cgtgggcaag accagcgcct acttcaactt cgcctttaag
     4201 ggcaacaaca aagagcagat tcacccgcac acactgttca cccctggcgc gccgggcggc
     4261 gacaagaccc acacctgccc cccctgccca gctccagaac tgctgggagg gccttccgtg
     4321 ttcctgttcc cccccaagcc caaggacacc ctgatgatca gcaggacccc cgaggtgacc
     4381 tgcgtggtgg tggccgtgtc ccacgaggac ccagaggtga agttcaactg gtacgtggac
     4441 ggcgtggagg tgcacaacgc caagaccaag cccagagagg agcagtacaa cagcacctac
     4501 agggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agaatacaag
     4561 tgcaaagtct ccaacaaggc cctggctgcc ccaatcgaaa agacaatcag caaggccaag
     4621 ggccagccac gggagcccca ggtgtacacc ctgcccccca gccgggagga gatgaccaag
     4681 aaccaggtgt ccctgacctg tctggtgaag ggcttctacc ccagcgatat cgccgtggag
     4741 tgggagagca acggccagcc cgagaacaac tacaagacca cccccccagt gctggacagc
     4801 gacggcagct tcttcctgta cagcaagctg accgtggaca agtccaggtg gcagcagggc
     4861 aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc
     4921 ctgagcctga gccccggcgg cagcggtctt aatgatattt tcgaggctca gaagattgag
     4981 tggcatgaac accaccacca tcaccactaa ggccggcctg tagtcactgg cacaacgcgt
     5041 tacccggtaa gccaatcggg tatacacggt cgtcatactg cagacagggt tcttctactt
     5101 tgcaagatag tcttgagtag taaaataaat agatagagaa aatcagcctc gactgtgcct
     5161 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     5221 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcacaaca ctcaacccta
     5281 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5341 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     5401 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     5461 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     5521 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     5581 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     5641 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     5701 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca
     5761 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg
     5821 aactaaacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     5881 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     5941 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     6001 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     6061 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     6121 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     6181 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     6241 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     6301 gccttcctgg agacatccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     6361 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     6421 ggtgcctgac acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     6481 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     6541 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     6601 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     6661 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     6721 taatcatggt cattaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6781 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6841 accatctggc cccagcgctg cgatgatacc gcgagaacca cgctcaccgg ctccggattt
     6901 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6961 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7021 tagtttgcgc aacgttgttg ccatcgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7081 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7141 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7201 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7261 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7321 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7381 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7441 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7501 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7561 aataagggcg acacggaaat gttgaatact catattcttc ctt
//
