LOCUS       sequence-302549-        6946 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 6946)
  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 6946)
  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-DLK1-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..6946
                     /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             4288..4332
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4333..4350
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4490..4507)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4496..4607
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4725..4745)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4730..5059
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4910..5045
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4972..4991
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5107..5154
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5173..5772
                     /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(5173..5192)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5669..5689
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5902..6023
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5939..5958)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5993..6012
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6076..6867,6868..6936))
                     /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     6699..6718
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6937..6946,1..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 tgaatgcttt
     2761 cctgcctgta atccccagaa cggcttctgc gaggacgaca atgtctgtag atgccagcct
     2821 ggatggcagg gccctctgtg tgatcagtgt gtgacatctc ctggctgcct gcacggactt
     2881 tgtggcgaac ctggccagtg tatctgcaca gatggatggg atggcgagct gtgcgataga
     2941 gatgtgcggg cctgttcttc tgccccttgc gccaacaata gaacctgcgt gtccctggac
     3001 gatggcctgt acgagtgtag ctgtgcccct ggctacagcg gcaaggactg tcagaaaaag
     3061 gacggcccct gcgtgatcaa tggctctcct tgtcagcacg gcggcacctg tgttgatgat
     3121 gaaggcagag ccagccacgc cagctgtctt tgtcctcctg gctttagcgg caatttctgc
     3181 gagatcgtgg ccaacagctg cacccctaat ccttgcgaga atgacggcgt gtgcacagac
     3241 atcggcggcg acttcagatg tagatgtcct gccggcttca tcgacaagac ctgcagcaga
     3301 cctgtgacca actgtgccag ctctccatgc cagaatggcg gcacatgtct gcagcacaca
     3361 caggtgtcct acgagtgcct gtgcaagcct gagtttaccg gcctgacctg cgtgaagaag
     3421 agggctctgt ctccccagca agtgacaaga ctgccttctg gctacggcct ggcctataga
     3481 cttacacctg gcgtgcacga actgccagtg cagcagcctg agcacagaat cctgaaggtg
     3541 tccatgaagg aactgaacaa gaaaacccct ctgctgaccg aaggccaagg cgcgccgggc
     3601 ggcgacaaga cccacacctg ccccccctgc ccagctccag aactgctggg agggccttcc
     3661 gtgttcctgt tcccccccaa gcccaaggac accctgatga tcagcaggac ccccgaggtg
     3721 acctgcgtgg tggtggccgt gtcccacgag gacccagagg tgaagttcaa ctggtacgtg
     3781 gacggcgtgg aggtgcacaa cgccaagacc aagcccagag aggagcagta caacagcacc
     3841 tacagggtgg tgtccgtgct gaccgtgctg caccaggact ggctgaacgg caaagaatac
     3901 aagtgcaaag tctccaacaa ggccctggct gccccaatcg aaaagacaat cagcaaggcc
     3961 aagggccagc cacgggagcc ccaggtgtac accctgcccc ccagccggga ggagatgacc
     4021 aagaaccagg tgtccctgac ctgtctggtg aagggcttct accccagcga tatcgccgtg
     4081 gagtgggaga gcaacggcca gcccgagaac aactacaaga ccaccccccc agtgctggac
     4141 agcgacggca gcttcttcct gtacagcaag ctgaccgtgg acaagtccag gtggcagcag
     4201 ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag
     4261 tccctgagcc tgagccccgg cggcagcggt cttaatgata ttttcgaggc tcagaagatt
     4321 gagtggcatg aacaccacca ccatcaccac taaggccggc ctgtagtcac tggcacaacg
     4381 cgttacccgg taagccaatc gggtatacac ggtcgtcata ctgcagacag ggttcttcta
     4441 ctttgcaaga tagtcttgag tagtaaaata aatagataga gaaaatcagc ctcgactgtg
     4501 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     4561 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcaca acactcaacc
     4621 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     4681 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     4741 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4801 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4861 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4921 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4981 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     5041 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     5101 gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg
     5161 aggaactaaa ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     5221 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     5281 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     5341 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     5401 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     5461 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     5521 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     5581 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     5641 cccgccttcc tggagacatc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     5701 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     5761 cccggtgcct gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg
     5821 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     5881 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     5941 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     6001 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     6061 gcgtaatcat ggtcattacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6121 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6181 cttaccatct ggccccagcg ctgcgatgat accgcgagaa ccacgctcac cggctccgga
     6241 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6301 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6361 taatagtttg cgcaacgttg ttgccatcgc tacaggcatc gtggtgtcac gctcgtcgtt
     6421 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6481 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6541 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6601 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6661 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6721 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6781 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6841 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6901 gggaataagg gcgacacgga aatgttgaat actcatattc ttcctt
//
