LOCUS       sequence-303035-        8320 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 8320)
  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 8320)
  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-TIE1-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8320
                     /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             5662..5706
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5707..5724
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5864..5881)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5870..5981
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6099..6119)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6104..6433
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6284..6419
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6346..6365
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6481..6528
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6547..7146
                     /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(6547..6566)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7043..7063
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7276..7397
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7313..7332)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7367..7386
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7450..8241,8242..8310))
                     /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     8073..8092
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8311..8320,1..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 cggccgccgc tgttgatctg
     2761 acactgctgg ctaacctgcg gctgaccgat cctcagagat tcttcctgac ctgtgtgtct
     2821 ggcgaagccg gcgctggaag aggatctgat gcttggggac ctcctctgct gctggaaaag
     2881 gacgacagaa tcgtgcggac acctcctgga cctccactga gactggccag aaatggcagc
     2941 caccaagtga ccctgagagg cttcagcaag ccttctgatc tcgtgggcgt gttctcttgt
     3001 gttggaggcg ctggcgctcg gagaaccaga gtgatctatg tgcacaacag ccctggcgct
     3061 catctgctgc ctgacaaagt gacccacacc gtgaacaagg gcgatacagc cgtgctgtcc
     3121 gccagagtgc acaaagaaaa gcagaccgac gtgatctgga agtccaacgg cagctacttc
     3181 tacaccctgg attggcacga ggcccaggat ggcagatttc tgctgcagct gcctaacgtg
     3241 cagcctccta gctctggcat ctacagcgcc acatacctgg aagcaagccc tctgggcagc
     3301 gccttcttca gactgatcgt tagaggatgc ggagccggca gatggggccc tggatgtaca
     3361 aaagaatgcc ctggctgtct gcacggcggc gtgtgtcacg atcatgatgg cgagtgtgtg
     3421 tgcccacctg gctttacagg caccagatgt gaacaggcct gcagagaggg cagattcggc
     3481 cagtcttgtc aagagcagtg tcccggcatc tctggctgca gaggcctgac cttttgcctg
     3541 cctgatcctt acggctgtag ctgtggatct ggatggcggg gctctcagtg tcaagaggct
     3601 tgtgcccctg gacactttgg cgctgattgc agactccagt gtcagtgcca gaacggcggc
     3661 acctgtgata gattcagcgg ctgtgtgtgt ccttctggct ggcatggcgt gcactgcgag
     3721 aagtccgata gaatccctca gatcctgaac atggccagcg agctggaatt caacctggaa
     3781 accatgcctc ggatcaactg cgccgctgcc ggaaatccat ttcctgtgcg gggatctatc
     3841 gagctgagaa agcctgatgg caccgtgctg ctgagcacca aggccatcgt ggaacctgag
     3901 aaaaccaccg ccgagttcga ggtgccaaga ctggttctgg ccgatagcgg cttctgggag
     3961 tgtagagtgt ctacctctgg cggccaggac agcagacggt tcaaagtgaa tgtgaaggtg
     4021 ccacctgtgc ctctggcagc ccctagactg ctgacaaaac agagcagaca gctggtggtg
     4081 tcccctctgg tgtcttttag cggagatggc cctatcagca cagtgcggct gcactacaga
     4141 ccccaggata gcaccatgga ctggtctacc attgtggtgg accccagcga gaacgtgaca
     4201 ctgatgaacc tgaggcctaa gaccggctac agcgtcagag ttcaactgag cagacctggc
     4261 gaaggcggag aaggcgcatg gggaccacct actctgatga ccacagattg ccctgagcct
     4321 ctgctccagc cttggctgga aggatggcat gtggaaggca ccgacagact gagagtgtct
     4381 tggtcactgc ctctggtgcc aggacctctc gttggagatg gctttctgct gagactgtgg
     4441 gatggcacaa gaggccaaga gcggagagag aatgtctcta gccctcaagc cagaactgcc
     4501 ctgctgacag gactgacacc tggcacacac taccagctgg atgtgcagct gtaccactgt
     4561 accctgctgg gacctgcttc tcctcctgct catgttctgc tgccaccatc tggaccacca
     4621 gcacctagac atctgcatgc ccaggctctg agcgatagcg agatccagct gacatggaag
     4681 cacccagaag ctctgcccgg accaatcagc aaatacgtgg tggaagtgca ggttgccggc
     4741 ggagctggcg atcctctgtg gattgatgtg gacagacccg aggaaaccag caccatcatc
     4801 aggggcctga acgccagcac cagatacctg ttcagaatgc gggcctctat ccaaggcctc
     4861 ggcgattggt ctaacaccgt ggaagagtct accctcggca acggactgca ggctgaagga
     4921 cctgtgcaag aaagcagagc cgccgaggaa ggcctggatc aaggcgcgcc gggcggcgac
     4981 aagacccaca cctgcccccc ctgcccagct ccagaactgc tgggagggcc ttccgtgttc
     5041 ctgttccccc ccaagcccaa ggacaccctg atgatcagca ggacccccga ggtgacctgc
     5101 gtggtggtgg ccgtgtccca cgaggaccca gaggtgaagt tcaactggta cgtggacggc
     5161 gtggaggtgc acaacgccaa gaccaagccc agagaggagc agtacaacag cacctacagg
     5221 gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaaaga atacaagtgc
     5281 aaagtctcca acaaggccct ggctgcccca atcgaaaaga caatcagcaa ggccaagggc
     5341 cagccacggg agccccaggt gtacaccctg ccccccagcc gggaggagat gaccaagaac
     5401 caggtgtccc tgacctgtct ggtgaagggc ttctacccca gcgatatcgc cgtggagtgg
     5461 gagagcaacg gccagcccga gaacaactac aagaccaccc ccccagtgct ggacagcgac
     5521 ggcagcttct tcctgtacag caagctgacc gtggacaagt ccaggtggca gcagggcaac
     5581 gtgttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg
     5641 agcctgagcc ccggcggcag cggtcttaat gatattttcg aggctcagaa gattgagtgg
     5701 catgaacacc accaccatca ccactaaggc cggcctgtag tcactggcac aacgcgttac
     5761 ccggtaagcc aatcgggtat acacggtcgt catactgcag acagggttct tctactttgc
     5821 aagatagtct tgagtagtaa aataaataga tagagaaaat cagcctcgac tgtgccttct
     5881 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     5941 actcccactg tcctttccta ataaaatgag gaaattgcat cacaacactc aaccctatct
     6001 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     6061 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     6121 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     6181 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     6241 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     6301 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     6361 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     6421 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt
     6481 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
     6541 taaaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     6601 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     6661 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     6721 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     6781 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     6841 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     6901 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt
     6961 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     7021 ttcctggaga catccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     7081 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     7141 gcctgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     7201 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     7261 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     7321 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     7381 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     7441 tcatggtcat taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7501 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7561 atctggcccc agcgctgcga tgataccgcg agaaccacgc tcaccggctc cggatttatc
     7621 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7681 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7741 tttgcgcaac gttgttgcca tcgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7801 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7861 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7921 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7981 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     8041 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     8101 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     8161 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     8221 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     8281 aagggcgaca cggaaatgtt gaatactcat attcttcctt
//
