LOCUS       sequence-303025-        8284 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 8284)
  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 8284)
  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-PTPRK-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8284
                     /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             5626..5670
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5671..5688
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5828..5845)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5834..5945
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6063..6083)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6068..6397
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6248..6383
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        6445..6492
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6511..7110
                     /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(6511..6530)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7007..7027
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7240..7361
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7277..7296)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7331..7350
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7414..8205,8206..8274))
                     /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     8037..8056
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8275..8284,1..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 tcaaggacaa
     2761 ttttctgccg gcggatgcac cttcgatgat ggacctggcg cctgcgacta ccaccaggac
     2821 ctgtacgatg acttcgagtg ggtgcacgtg tccgctcaag agcctcacta tctgcctcca
     2881 gagatgcccc agggcagcta catgatcgtg gacagcagcg atcacgaccc tggcgagaaa
     2941 gctagactgc agctgcccac catgaaggaa aacgacaccc actgcatcga cttcagctac
     3001 ctgctgtact cccagaaggg actgaacccc ggcacactga acatcctcgt gcgcgtgaac
     3061 aagggccctc tggccaatcc tatctggaac gtgaccggct tcaccggcag agattggctg
     3121 agagccgaac tggccgtgtc caccttctgg cccaatgagt accaagtgat cttcgaggcc
     3181 gaggtgtccg gcggcagatc tggatatatc gccatcgacg acatccaggt gctgagctac
     3241 ccctgcgata agagccctca cttcctgaga ctgggcgacg tggaagtgaa cgccggacag
     3301 aatgccacct tccagtgtat cgccacaggc agggatgccg tgcacaacaa actgtggctg
     3361 cagcggagaa acggcgagga cattcctgtg gctcagacca agaacatcaa ccacagaaga
     3421 ttcgccgcca gcttccggct gcaagaagtg accaagaccg accaggatct gtatagatgc
     3481 gtgacccaga gcgagagagg cagcggcgtt agcaatttcg cccagctgat tgtgcgcgag
     3541 cctcctagac ctatcgctcc tccacagctg cttggcgtgg gcccaacata tctgctgatc
     3601 cagctgaacg ccaacagcat catcggcgac ggccccatca tcctgaaaga ggtggaatac
     3661 cggatgacca gcggcagctg gacagaaacc catgccgtga atgcccctac ctacaagctg
     3721 tggcatctgg accccgacac cgagtacgag atcagagtgc tgctgaccag acctggcgaa
     3781 ggcggaacag gacttcctgg acctcctctg atcacccgga ccaagtgtgc cgagcctatg
     3841 cggacaccca agacactgaa gatcgccgag attcaggcca gacggatcgc cgtggattgg
     3901 gagagcctgg gctacaacat cacccggtgc cacaccttca acgtgaccat ctgctaccac
     3961 tacttccggg gccacaacga gagcaaggcc gactgccttg acatggaccc taaggctcct
     4021 cagcacgtgg tcaaccatct gccaccttac accaacgtgt ccctgaagat gatcctgaca
     4081 aaccccgagg gccgcaaaga gagcgaggaa accatcatcc agaccgacga ggacgtgcca
     4141 ggaccagtgc ctgtgaaatc tctgcagggc accagcttcg agaacaagat cttcctgaat
     4201 tggaaagagc cactggaccc caacggaatc atcacccagt atgagatctc ttacagcagc
     4261 atccgcagct tcgaccctgc tgtgcctgtt gctggaccac ctcagaccgt gtccaacctg
     4321 tggaatagca cccaccacgt gttcatgcat ctgcaccctg gcaccaccta ccagttcttc
     4381 atcagagcca gcaccgtgaa aggcttcgga cctgccacag ccatcaatgt gactaccaac
     4441 atcagcgccc ctacactgcc cgactatgaa ggcgttgacg ccagcctgaa tgagacagcc
     4501 accaccatta ccgtgctgct gcgacctgct caggctaaag gcgcccctat cagcgcctat
     4561 cagatcgtgg tggaagaact gcacccacac aggaccaagc gagaagccgg cgctatggaa
     4621 tgctaccagg tgccagtgac ctaccagaac gccatgtctg gcggagcccc ttactatttt
     4681 gccgccgaac tgcctcctgg caacctgcct gaacctgctc cttttaccgt gggcgacaac
     4741 cggacctacc aaggcttctg gaatcctcca ctggctcccc ggaagggcta taacatctac
     4801 ttccaagcca tgagcagcgt cgagaaagaa acaaagaccc agtgcgtgcg gatcgccaca
     4861 aaagccgcta ccgaggaacc cgaagtgatc cccgatcctg ccaagcagac cgacagagtg
     4921 gtcaaaggcg cgccgggcgg cgacaagacc cacacctgcc ccccctgccc agctccagaa
     4981 ctgctgggag ggccttccgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc
     5041 agcaggaccc ccgaggtgac ctgcgtggtg gtggccgtgt cccacgagga cccagaggtg
     5101 aagttcaact ggtacgtgga cggcgtggag gtgcacaacg ccaagaccaa gcccagagag
     5161 gagcagtaca acagcaccta cagggtggtg tccgtgctga ccgtgctgca ccaggactgg
     5221 ctgaacggca aagaatacaa gtgcaaagtc tccaacaagg ccctggctgc cccaatcgaa
     5281 aagacaatca gcaaggccaa gggccagcca cgggagcccc aggtgtacac cctgcccccc
     5341 agccgggagg agatgaccaa gaaccaggtg tccctgacct gtctggtgaa gggcttctac
     5401 cccagcgata tcgccgtgga gtgggagagc aacggccagc ccgagaacaa ctacaagacc
     5461 acccccccag tgctggacag cgacggcagc ttcttcctgt acagcaagct gaccgtggac
     5521 aagtccaggt ggcagcaggg caacgtgttc agctgcagcg tgatgcacga ggccctgcac
     5581 aaccactaca cccagaagtc cctgagcctg agccccggcg gcagcggtct taatgatatt
     5641 ttcgaggctc agaagattga gtggcatgaa caccaccacc atcaccacta aggccggcct
     5701 gtagtcactg gcacaacgcg ttacccggta agccaatcgg gtatacacgg tcgtcatact
     5761 gcagacaggg ttcttctact ttgcaagata gtcttgagta gtaaaataaa tagatagaga
     5821 aaatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     5881 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     5941 gcatcacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     6001 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     6061 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     6121 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     6181 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     6241 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     6301 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     6361 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     6421 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata
     6481 gtataatacg acaaggtgag gaactaaacc atgaccgagt acaagcccac ggtgcgcctc
     6541 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     6601 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     6661 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     6721 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     6781 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     6841 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     6901 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     6961 gccgagcgcg ccggggtgcc cgccttcctg gagacatccg cgccccgcaa cctccccttc
     7021 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     7081 tggtgcatga cccgcaagcc cggtgcctga cacgtgctac gagatttcga ttccaccgcc
     7141 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     7201 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     7261 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     7321 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     7381 acctctagct agagcttggc gtaatcatgg tcattaccaa tgcttaatca gtgaggcacc
     7441 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7501 aactacgata cgggagggct taccatctgg ccccagcgct gcgatgatac cgcgagaacc
     7561 acgctcaccg gctccggatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7621 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     7681 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccatcgcta caggcatcgt
     7741 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7801 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7861 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7921 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7981 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     8041 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     8101 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     8161 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     8221 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatattctt
     8281 cctt
//
