LOCUS       sequence-302937-        7795 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 7795)
  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 7795)
  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-LRIT3-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..7795
                     /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     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             5137..5181
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5182..5199
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5339..5356)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5345..5456
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5574..5594)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5579..5908
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5759..5894
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5821..5840
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5956..6003
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6022..6621
                     /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(6022..6041)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6518..6538
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6751..6872
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6788..6807)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6842..6861
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6925..7716,7717..7785))
                     /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     7548..7567
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7786..7795,1..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 cggccgcctt gtgtcctagc
     2761 cagtgtacat gtgactacca cggcagaaac gacggctctg gaagcagact ggtgctgtgc
     2821 aacgacatgg acatgaacga gctgcccacc aacctgcctg tggacaccgt gaagctgcgg
     2881 atcgagaaaa ccgtgatcag aagaatcagc gccgaggcct tctactacct ggtggaactg
     2941 cagtacctgt gggtcaccta caacagcgtg gccagcatcg accccagcag cttctacaat
     3001 ctgaagcagc tgcacgagct gcggctggat ggaaattctc tggctgcctt tccttgggcc
     3061 tctctgctgg atatgcctct gctgagaacc ctggacctgc acaacaacaa gatcaccagc
     3121 gtgcccaacg aggccctgag atacctgaag aacctggcct acctggatct gagcagcaac
     3181 agactgacaa ccctgcctcc tgacttcctg gaaagctgga cccacctggt gtctacacct
     3241 agcggagtgc tggatctgtc ccctagcaga atcatcctgg gcctgcagga caacccctgg
     3301 ttctgcgatt gccacatcag caagatgatc gagctgagca aggtggtgga ccctgctatt
     3361 gtgctgctgg accctctgat gacctgcagc gagcctgaga gactgaccgg cattctgttc
     3421 cagagagccg agctggaaca ctgcctgaag cctagcgtga tgaccagcgc cacaaagatc
     3481 atgagcgccc tgggctctaa cgtgctgctg agatgtgatg ccaccggctt tcccacacct
     3541 cagatcacct ggaccagatc cgacagcagc cccgtgaact acaccgtgat ccaagagtct
     3601 cccgaagagg gcgtcagatg gtccattatg agcctgacag gcatcagcag caaggacgcc
     3661 ggcgattaca agtgcaaggc caagaatctg gccggcatgt ctgaggccgt ggtcacagtg
     3721 acagtgctgg gcatcaccac cacacctatt cctccagaca ccagcgagag aaccggcgat
     3781 catcctgaat gggatgtgca gcctggcagc ggcagatcta catctgtgtc tagcgccagc
     3841 tcctacctgt ggtctagcag ctttagcccc acctccagct tttccgccag cacactgtct
     3901 ccaccaagta ccgcaagctt cagtctgagc cctttcagca gcagcaccgt gtcctctacc
     3961 acaacactga gcaccagcat cagcgccagc accaccatgg ccaacaagag aagctttcag
     4021 ctgcaccaag gcggcaagag aaacctgaag gtggccaaga acggcagcaa gctgcctcca
     4081 gccagcacct ccaaaaaaga ggaactggcc ctgctggatc agaccatgct gacagagaca
     4141 aacgccgcca tcgagaacct gagagtggtg tccgagacaa aagaaagcgt gaccctgacc
     4201 tggaacatga tcaacaccac acacaacagc gccgtgaccg tgctgtactc taagtacggc
     4261 ggaaaggacc tgctgctcct gaacgccgac agctccaaga atcaagtgac catcgacgga
     4321 ctggaacctg gcggccagta tatggcctgt gtgtgtccta aaggcgtgcc acctcagaaa
     4381 gaccagtgca tcaccttcag caccgagaga gtggaaggcg acgacagcca gtggtctggc
     4441 gcgccgggcg gcgacaagac ccacacctgc cccccctgcc cagctccaga actgctggga
     4501 gggccttccg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagcaggacc
     4561 cccgaggtga cctgcgtggt ggtggccgtg tcccacgagg acccagaggt gaagttcaac
     4621 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agcccagaga ggagcagtac
     4681 aacagcacct acagggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc
     4741 aaagaataca agtgcaaagt ctccaacaag gccctggctg ccccaatcga aaagacaatc
     4801 agcaaggcca agggccagcc acgggagccc caggtgtaca ccctgccccc cagccgggag
     4861 gagatgacca agaaccaggt gtccctgacc tgtctggtga agggcttcta ccccagcgat
     4921 atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccca
     4981 gtgctggaca gcgacggcag cttcttcctg tacagcaagc tgaccgtgga caagtccagg
     5041 tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac
     5101 acccagaagt ccctgagcct gagccccggc ggcagcggtc ttaatgatat tttcgaggct
     5161 cagaagattg agtggcatga acaccaccac catcaccact aaggccggcc tgtagtcact
     5221 ggcacaacgc gttacccggt aagccaatcg ggtatacacg gtcgtcatac tgcagacagg
     5281 gttcttctac tttgcaagat agtcttgagt agtaaaataa atagatagag aaaatcagcc
     5341 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     5401 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcacaa
     5461 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     5521 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     5581 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     5641 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     5701 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     5761 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     5821 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     5881 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     5941 atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac
     6001 gacaaggtga ggaactaaac catgaccgag tacaagccca cggtgcgcct cgccacccgc
     6061 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     6121 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     6181 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     6241 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     6301 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     6361 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     6421 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     6481 gccggggtgc ccgccttcct ggagacatcc gcgccccgca acctcccctt ctacgagcgg
     6541 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     6601 acccgcaagc ccggtgcctg acacgtgcta cgagatttcg attccaccgc cgccttctat
     6661 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     6721 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     6781 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     6841 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     6901 tagagcttgg cgtaatcatg gtcattacca atgcttaatc agtgaggcac ctatctcagc
     6961 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7021 acgggagggc ttaccatctg gccccagcgc tgcgatgata ccgcgagaac cacgctcacc
     7081 ggctccggat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     7141 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     7201 ttcgccagtt aatagtttgc gcaacgttgt tgccatcgct acaggcatcg tggtgtcacg
     7261 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7321 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7381 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7441 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     7501 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     7561 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     7621 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7681 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7741 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatattct tcctt
//
