LOCUS       sequence-484529-        8128 bp    DNA     circular SYN 25-AUG-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 8128)
  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 8128)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 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-PTK7-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..8128
                     /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"
     RBS             4746..4754
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             5470..5514
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5515..5532
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5672..5689)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5678..5789
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5907..5927)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5912..6241
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6092..6227
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6154..6173
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6289..6336
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6355..6954
                     /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(6355..6374)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6851..6871
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7084..7205
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7121..7140)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7175..7194
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7258..8049,8050..8118))
                     /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     7881..7900
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8119..8128,1..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 tagsccacca 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 catcgtgttt
     2761 atcaagcagc ctagcagcca ggacgccctg caaggccgta gagcactgct gagatgcgaa
     2821 gtggaagccc ctggacctgt gcacgtttac tggttgcttg atggcgcccc tgtgcaggac
     2881 accgagagaa gatttgccca gggcagcagc ctgtctttcg ccgctgttga cagactgcag
     2941 gacagcggca cctttcagtg cgtggccaga gatgatgtga ccggcgagga agccagaagc
     3001 gccaacgcca gcttcaacat caagtggatc gaggctggcc ccgtggtgct gaaacatcct
     3061 gcctctgagg ccgagatcca gccacagaca caagtgaccc tgcggtgcca catcgacgga
     3121 caccctagac caacctacca gtggttcaga gatggcaccc ctctgtccga cggccagagc
     3181 aatcataccg tgtccagcaa agagcggaac ctgacactga ggcctgccgg acctgaacac
     3241 agcggcctgt attcttgttg tgcccacagc gcctttggcc aggcctgtag ctctcagaac
     3301 ttcaccctga gcattgccga cgagagcttc gccagagttg tgctggctcc tcaggatgtg
     3361 gtggtggcca gatacgaaga ggccatgttc cactgccagt tcagcgctca gcctcctcca
     3421 tctctgcagt ggctgttcga ggacgagaca cccatcacca acagaagccg gcctccacat
     3481 ctgagaaggg ccaccgtgtt tgccaatggc agcctgctgc tgactcaagt gcggcctaga
     3541 aacgccggca tctacagatg tatcggccag ggccaaagag gccctcctat catcctggaa
     3601 gccacactgc acctggccga gattgaggac atgcccctgt tcgagcccag agtgtttaca
     3661 gccggcagcg aggaaagagt gacctgcctg cctcctaagg gactgcctga gccttctgtt
     3721 tggtgggaac acgctggcgt cagactgcct acacacggca gagtgtacca gaaaggccac
     3781 gaactggtgc tggccaatat cgccgaatcc gacgcaggcg tgtacacatg tcatgccgcc
     3841 aatctggccg gacagagaag gcaggacgtg aacatcacag tggccacagt gcccagctgg
     3901 ctgaagaagc ctcaggactc tcagctggaa gagggcaagc ctggctacct ggattgtctg
     3961 acacaggcca cacctaagcc taccgtcgtg tggtacagaa accagatgct gatctccgag
     4021 gacagcagat tcgaggtgtt caagaacggc accctgcgga tcaacagcgt ggaagtgtac
     4081 gacggcacct ggtacagatg catgagcagc acaccagccg gctccattga agcccaggct
     4141 agagtgcagg tcctggaaaa gctgaagttc acccctccac ctcagccaca gcagtgcatg
     4201 gaattcgaca aagaggctac agtcccctgc agcgccacag gcagagagaa gcctacaatc
     4261 aagtgggaga gagccgacgg cagctccctg cctgaatggg tcacagataa tgccggcact
     4321 ctgcactttg ccagagtgac cagggacgac gccggcaatt atacctgtat cgccagcaat
     4381 ggcccacagg gccagattag agcccacgtg cagctgacag tggccgtgtt catcaccttc
     4441 aaggtggaac ccgagcggac caccgtgtat cagggacata cagctctgct gcagtgcgag
     4501 gctcagggcg atcctaagcc tctgatccag tggaagggca aagaccggat tctggacccc
     4561 acaaagctgg gccccagaat gcacatcttc cagaacggct ccctcgtgat ccacgatgtg
     4621 gcccctgaag atagcggccg gtacacctgt attgccggaa acagctgcaa tatcaagcac
     4681 acagaggccc ctctgtacgt ggtggacaag cctgtgcctg aggaatctga aggccctggc
     4741 agcccacctc cttacaagat gatccagaca ggcgcgccgg gcggcgacaa gacccacacc
     4801 tgccccccct gcccagctcc agaactgctg ggagggcctt ccgtgttcct gttccccccc
     4861 aagcccaagg acaccctgat gatcagcagg acccccgagg tgacctgcgt ggtggtggcc
     4921 gtgtcccacg aggacccaga ggtgaagttc aactggtacg tggacggcgt ggaggtgcac
     4981 aacgccaaga ccaagcccag agaggagcag tacaacagca cctacagggt ggtgtccgtg
     5041 ctgaccgtgc tgcaccagga ctggctgaac ggcaaagaat acaagtgcaa agtctccaac
     5101 aaggccctgg ctgccccaat cgaaaagaca atcagcaagg ccaagggcca gccacgggag
     5161 ccccaggtgt acaccctgcc ccccagccgg gaggagatga ccaagaacca ggtgtccctg
     5221 acctgtctgg tgaagggctt ctaccccagc gatatcgccg tggagtggga gagcaacggc
     5281 cagcccgaga acaactacaa gaccaccccc ccagtgctgg acagcgacgg cagcttcttc
     5341 ctgtacagca agctgaccgt ggacaagtcc aggtggcagc agggcaacgt gttcagctgc
     5401 agcgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgag cctgagcccc
     5461 ggcggcagcg gtcttaatga tattttcgag gctcagaaga ttgagtggca tgaacaccac
     5521 caccatcacc actaaggccg gcctgtagtc actggcacaa cgcgttaccc ggtaagccaa
     5581 tcgggtatac acggtcgtca tactgcagac agggttcttc tactttgcaa gatagtcttg
     5641 agtagtaaaa taaatagata gagaaaatca gcctcgactg tgccttctag ttgccagcca
     5701 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     5761 ctttcctaat aaaatgagga aattgcatca caacactcaa ccctatctcg gtctattctt
     5821 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     5881 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     5941 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     6001 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     6061 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     6121 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     6181 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     6241 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa
     6301 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatgacc
     6361 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     6421 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     6481 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     6541 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     6601 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     6661 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     6721 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     6781 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagaca
     6841 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     6901 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacacgtg
     6961 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     7021 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     7081 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     7141 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     7201 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatta
     7261 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     7321 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     7381 cgctgcgatg ataccgcgag aaccacgctc accggctccg gatttatcag caataaacca
     7441 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     7501 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     7561 tgttgccatc gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     7621 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     7681 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7741 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7801 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7861 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7921 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7981 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     8041 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     8101 gaaatgttga atactcatat tcttcctt
//
