LOCUS       sequence-302907-        7657 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 7657)
  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 7657)
  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-FLT3-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..7657
                     /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             4999..5043
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5044..5061
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5201..5218)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5207..5318
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5436..5456)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5441..5770
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5621..5756
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5683..5702
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5818..5865
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5884..6483
                     /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(5884..5903)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6380..6400
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6613..6734
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6650..6669)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6704..6723
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6787..7578,7579..7647))
                     /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     7410..7429
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7648..7657,1..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 cggccgccaa tcaagacctg
     2761 cctgtgatta agtgcgtgct gatcaaccac aagaacaacg acagcagcgt gggcaagagc
     2821 agcagctacc ctatggtgtc tgagagccct gaggatctgg gctgtgccct cagacctcag
     2881 tctagcggca cagtgtatga ggccgctgct gtggaagtgg atgtgtccgc cagcatcaca
     2941 ctgcaggtcc tggttgatgc ccctggcaac atcagctgcc tgtgggtgtt caagcacagc
     3001 agcctgaact gccagcctca cttcgacctg cagaacagag gcgtggtgtc catggtcatc
     3061 ctgaagatga ccgagacaca ggccggcgag tacctgctgt ttatccagag cgaggccacc
     3121 aactacacca tcctgttcac cgtgtccatc cggaacaccc tgctgtacac cctgagaagg
     3181 ccctacttcc gcaagatgga aaaccaggac gccctcgtgt gcatcagcga gtctgtgcct
     3241 gagcctatcg tggaatgggt gctgtgtgat agccagggcg agagctgcaa agaggaatct
     3301 cctgccgtcg tgaagaaaga ggaaaaggtc ctgcacgagc tgttcggcac cgacatcaga
     3361 tgctgcgcca gaaacgagct gggcagagag tgcaccagac tgttcaccat cgacctgaat
     3421 cagaccccac agacaaccct gcctcagctg ttcctgaaag tgggcgagcc cctgtggatc
     3481 agatgcaaag ccgtgcacgt gaaccacggc ttcggactga catgggaact cgagaacaag
     3541 gccctggaag agggcaacta cttcgagatg agcacctaca gcaccaaccg gaccatgatc
     3601 agaatcctgt tcgccttcgt gtccagcgtg gccagaaatg acaccggcta ctacacctgt
     3661 agcagcagca agcaccctag ccagtctgcc ctggtcacca tcgtggaaaa gggcttcatc
     3721 aacgccacca acagcagcga ggactacgag atcgaccagt acgaggaatt ctgcttcagc
     3781 gtgcggttca aggcctatcc tcagatccgg tgcacctgga ccttcagcag aaagagcttc
     3841 ccctgcgagc agaaaggcct ggacaacggc tacagcatca gcaagttctg caaccacaaa
     3901 caccagcctg gcgagtacat cttccacgcc gagaacgacg atgcccagtt caccaagatg
     3961 ttcaccctga acatccggcg gaagcctcag gtgctggctg aagcctctgc cagccaggcc
     4021 agctgttttt ccgatggcta ccctctgcct agctggacct ggaagaagtg cagcgacaag
     4081 agccccaact gcaccgaaga gatcaccgaa ggcgtgtgga accggaaggc caacagaaag
     4141 gtgttcggcc agtgggtgtc cagcagcacc ctgaatatga gcgaggctat caagggcttc
     4201 ctggtcaagt gctgcgccta caatagcctg ggcaccagct gcgagacaat cctgctgaat
     4261 agccccggac catttccatt catccaggac aacatctctg gcgcgccggg cggcgacaag
     4321 acccacacct gccccccctg cccagctcca gaactgctgg gagggccttc cgtgttcctg
     4381 ttccccccca agcccaagga caccctgatg atcagcagga cccccgaggt gacctgcgtg
     4441 gtggtggccg tgtcccacga ggacccagag gtgaagttca actggtacgt ggacggcgtg
     4501 gaggtgcaca acgccaagac caagcccaga gaggagcagt acaacagcac ctacagggtg
     4561 gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagaata caagtgcaaa
     4621 gtctccaaca aggccctggc tgccccaatc gaaaagacaa tcagcaaggc caagggccag
     4681 ccacgggagc cccaggtgta caccctgccc cccagccggg aggagatgac caagaaccag
     4741 gtgtccctga cctgtctggt gaagggcttc taccccagcg atatcgccgt ggagtgggag
     4801 agcaacggcc agcccgagaa caactacaag accacccccc cagtgctgga cagcgacggc
     4861 agcttcttcc tgtacagcaa gctgaccgtg gacaagtcca ggtggcagca gggcaacgtg
     4921 ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc
     4981 ctgagccccg gcggcagcgg tcttaatgat attttcgagg ctcagaagat tgagtggcat
     5041 gaacaccacc accatcacca ctaaggccgg cctgtagtca ctggcacaac gcgttacccg
     5101 gtaagccaat cgggtataca cggtcgtcat actgcagaca gggttcttct actttgcaag
     5161 atagtcttga gtagtaaaat aaatagatag agaaaatcag cctcgactgt gccttctagt
     5221 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     5281 cccactgtcc tttcctaata aaatgaggaa attgcatcac aacactcaac cctatctcgg
     5341 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5401 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     5461 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     5521 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     5581 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     5641 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     5701 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     5761 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt
     5821 gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa
     5881 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     5941 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     6001 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     6061 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     6121 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     6181 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     6241 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     6301 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     6361 ctggagacat ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     6421 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     6481 tgacacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     6541 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     6601 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     6661 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     6721 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     6781 tggtcattac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6841 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6901 tggccccagc gctgcgatga taccgcgaga accacgctca ccggctccgg atttatcagc
     6961 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     7021 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     7081 gcgcaacgtt gttgccatcg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     7141 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7201 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7261 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7321 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7381 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7441 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7501 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7561 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7621 ggcgacacgg aaatgttgaa tactcatatt cttcctt
//
