LOCUS       sequence-302991-        8110 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of cell-surface protein partial 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 8110)
  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 8110)
  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-NOTCH4(Trunc2)-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8110
                     /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             5452..5496
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5497..5514
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5654..5671)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5660..5771
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5889..5909)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5894..6223
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6074..6209
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6136..6155
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6271..6318
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6337..6936
                     /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(6337..6356)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6833..6853
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7066..7187
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7103..7122)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7157..7176
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7240..8031,8032..8100))
                     /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     7863..7882
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8101..8110,1..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 cggccgccag aggactgctg
     2761 tgtggatctt ttccagagcc ttgcgccaat ggcggcacat gtctgtctct gtctctcggc
     2821 cagggaacct gtcagtgtgc tcctggattt ctgggcgaga cttgtcagtt ccccgatcct
     2881 tgtcagaacg cccagctgtg tcagaatggc ggctcttgtc aagctctgct gcctgctcct
     2941 ctgggactgc cttcttctcc atctccactg acacccagct ttctgtgcac ctgtctgcct
     3001 ggctttaccg gcgaaagatg ccaggccaag ctggaagatc cctgtcctcc tagcttctgc
     3061 agcaagaggg gcagatgtca catccaggca agcggcagac ctcagtgttc ttgtatgcct
     3121 ggctggacag gcgagcagtg ccagctgaga gatttctgca gcgccaatcc ttgtgtgaac
     3181 ggcggcgtgt gtctggccac atatcctcag atccagtgcc actgtcctcc aggctttgag
     3241 ggacacgcct gcgagaggga tgtgaacgag tgttttcagg accccggacc ttgtcctaag
     3301 ggcacctctt gtcacaacac cctgggcagc ttccagtgtc tgtgtcctgt gggacaagag
     3361 ggacccagat gtgaactgag agccgggcct tgtccaccta gaggctgttc taatggcggg
     3421 acctgtcagc tgatgcccga gaaggatagc accttccacc tgtgtctgtg cccacctggc
     3481 ttcatcggcc ccgattgcga agtgaacccc gacaattgtg tgtcccacca gtgccagaac
     3541 ggcggaactt gtcaggacgg cctggacacc tatacctgcc tgtgtccaga gacatggacc
     3601 ggctgggatt gcagcgagga tgtggatgag tgtgaaacac agggccctcc acactgcaga
     3661 aacggcggga catgccagaa tagcgccggc tcttttcact gcgtgtgtgt gtcaggctgg
     3721 ggcggcacaa gctgcgagga aaatctggac gattgcattg ccgccacatg cgcccctggc
     3781 agcacatgta ttgatagagt gggctccttc agctgtctct gccctcctgg aagaacaggc
     3841 ctgctgtgtc acctggaaga tatgtgcctg agccagcctt gtcatggcga cgcccagtgt
     3901 agcacaaatc ctctgaccgg cagcaccctg tgtctttgtc agcctggcta ctctggccct
     3961 acctgccacc aggatctgga tgaatgtctg atggcccagc agggcccttc tccttgtgaa
     4021 catggcggaa gctgtctgaa cacacccggc agcttcaact gcttgtgtcc tcctggctac
     4081 accggaagca gatgcgaggc cgatcacaac gaatgcctgt ctcagccttg ccatcctggc
     4141 tctacctgtc tggatctgct ggccaccttt cactgcctct gtccgcctgg actggaagga
     4201 cagctgtgcg aggtggaaac aaatgagtgc gccagcgctc cttgcctgaa tcacgccgat
     4261 tgccacgacc tgctgaacgg ctttcagtgt atctgcctgc ctggattcag cggcaccaga
     4321 tgcgaagagg acatcgacga gtgtagaagc agcccttgtg ccaacggtgg ccagtgtcaa
     4381 gatcagccag gcgccttcca ctgcaagtgt ctgccaggat ttgaaggccc cagatgccag
     4441 accgaagtgg acgagtgtct gagcgatcca tgtccagtgg gagcctcttg tcttgacctg
     4501 cctggcgcct tcttttgtct ttgccctagc ggctttacag ggcagctgtg tgaagtgccc
     4561 ctgtgcgctc ctaatctgtg ccagcctaaa cagatctgca aggaccagaa ggataaggcc
     4621 aactgccttt gtcctgatgg ctctccagga tgcgccccac ctgaggataa ttgcacatgt
     4681 caccacggcc actgccagag aagctcttgt gtgtgtgatg tcggatggac aggacccgag
     4741 tgcgaagccg aaggcgcgcc gggcggcgac aagacccaca cctgcccccc ctgcccagct
     4801 ccagaactgc tgggagggcc ttccgtgttc ctgttccccc ccaagcccaa ggacaccctg
     4861 atgatcagca ggacccccga ggtgacctgc gtggtggtgg ccgtgtccca cgaggaccca
     4921 gaggtgaagt tcaactggta cgtggacggc gtggaggtgc acaacgccaa gaccaagccc
     4981 agagaggagc agtacaacag cacctacagg gtggtgtccg tgctgaccgt gctgcaccag
     5041 gactggctga acggcaaaga atacaagtgc aaagtctcca acaaggccct ggctgcccca
     5101 atcgaaaaga caatcagcaa ggccaagggc cagccacggg agccccaggt gtacaccctg
     5161 ccccccagcc gggaggagat gaccaagaac caggtgtccc tgacctgtct ggtgaagggc
     5221 ttctacccca gcgatatcgc cgtggagtgg gagagcaacg gccagcccga gaacaactac
     5281 aagaccaccc ccccagtgct ggacagcgac ggcagcttct tcctgtacag caagctgacc
     5341 gtggacaagt ccaggtggca gcagggcaac gtgttcagct gcagcgtgat gcacgaggcc
     5401 ctgcacaacc actacaccca gaagtccctg agcctgagcc ccggcggcag cggtcttaat
     5461 gatattttcg aggctcagaa gattgagtgg catgaacacc accaccatca ccactaaggc
     5521 cggcctgtag tcactggcac aacgcgttac ccggtaagcc aatcgggtat acacggtcgt
     5581 catactgcag acagggttct tctactttgc aagatagtct tgagtagtaa aataaataga
     5641 tagagaaaat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     5701 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     5761 gaaattgcat cacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     5821 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5881 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     5941 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     6001 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     6061 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     6121 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     6181 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     6241 tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc
     6301 ggcatagtat aatacgacaa ggtgaggaac taaaccatga ccgagtacaa gcccacggtg
     6361 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     6421 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     6481 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     6541 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     6601 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     6661 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     6721 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     6781 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga catccgcgcc ccgcaacctc
     6841 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     6901 cgcacctggt gcatgacccg caagcccggt gcctgacacg tgctacgaga tttcgattcc
     6961 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     7021 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     7081 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     7141 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     7201 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat taccaatgct taatcagtga
     7261 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7321 gtagataact acgatacggg agggcttacc atctggcccc agcgctgcga tgataccgcg
     7381 agaaccacgc tcaccggctc cggatttatc agcaataaac cagccagccg gaagggccga
     7441 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7501 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca tcgctacagg
     7561 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7621 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7681 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7741 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7801 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7861 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7921 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7981 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     8041 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     8101 attcttcctt
//
