LOCUS       sequence-458136-        7477 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of cell-surface protein extracellular domain 
            fused to COMP5AP-AviTag-9xHis. Protein is secreted from cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7477)
  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 7477)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-THY1-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..7477
                     /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             3127..3150
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4810..4854
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             4855..4881
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(5021..5038)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5027..5138
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5256..5276)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5261..5590
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5441..5576
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5503..5522
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5638..5685
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5704..6303
                     /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(5704..5723)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6200..6220
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6433..6554
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6470..6489)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6524..6543
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6607..7398,7399..7467))
                     /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     7230..7249
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7468..7477,1..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 cggccgccca gaaagtgaca
     2761 tctctgacag cttgcctggt ggaccagagc ctgagactgg actgcagaca cgagaatacc
     2821 agcagcagcc ccatccagta cgagttcagc ctgaccagag aaacaaagaa acacgtgctg
     2881 ttcggcaccg tgggcgtgcc agagcacaca tacagaagca ggaccaactt caccagcaag
     2941 tacaacatga aggtgctgta cctgagcgcc ttcacctcca aggatgaggg cacatacaca
     3001 tgcgccctgc accactctgg acacagccct ccaatcagca gccagaacgt gaccgtgctg
     3061 cgggacaaac ttggcgcgcc ggaagatcaa gttgatccaa gattgattga tggcaagggc
     3121 ggatctctgg aggtgctgtt ccagggacca ggcggaagcg gcaatcccgg cggcgatctg
     3181 gccccccaga tgctgcgcga gctgcaggag accaatgccg ccctgcagga cgtgcgcgag
     3241 ctgctgcgcc accgcgtgaa ggagatcacc ttcctgaaga acaccgtgat ggagtgcgac
     3301 gcctgcggta tgcagccctc tagaggctcc ggctccatca tcccagttga ggaggagaac
     3361 ccggacttct ggaaccgcga ggcagccgag gccctgggtg ccgccaagaa gctgcagcct
     3421 gcacagacag ccgccaagaa cctcatcatc ttcctgggcg atgggatggg ggtgtctacg
     3481 gtgacagctg ccaggatcct aaaagggcag aagaaggaca aactggggcc tgagataccc
     3541 ctggccatgg accgcttccc atatgtggct ctgtccaaga catacaatgt agacaaacat
     3601 gtgccagaca gtggagccac agccacggcc tacctgtgcg gggtcaaggg caacttccag
     3661 accattggct tgagtgcagc cgcccgcttt aaccagtgca acacgacacg cggcaacgag
     3721 gtcatctccg tgatgaatcg ggccaagaaa gcagggaagt cagtgggagt ggtaaccacc
     3781 acacgagtgc agcacgcctc gccagccggc acctacgccc acacggtgaa ccgcaactgg
     3841 tactcggacg ccgacgtgcc tgcctcggcc cgccaggagg ggtgccagga catcgctacg
     3901 cagctcatct ccaacatgga cattgacgtg atcctaggtg gaggccgaaa gtacatgttt
     3961 cgcatgggaa ccccagaccc tgagtaccca gatgactaca gccaaggtgg gaccaggctg
     4021 gacgggaaga atctggtgca ggaatggctg gcgaagcgcc agggtgcccg gtatgtgtgg
     4081 aaccgcactg agctcatgca ggcttccctg gacccgtctg tgacccatct catgggtctc
     4141 tttgagcctg gagacatgaa atacgagatc caccgagact ccacactgga cccctccctg
     4201 atggagatga cagaggctgc cctgcgcctg ctgagcagga acccccgcgg cttcttcctc
     4261 ttcgtggagg gtggtcgcat cgaccatggt catcatgaaa gcagggctta ccgggcactg
     4321 actgagacga tcatgttcga cgacgccatt gagagggcgg gccagctcac cagcgaggag
     4381 gacacgctga gcctcgtcac tgccgaccac tcccacgtct tctccttcgg aggctacccc
     4441 ctgcgaggga gctccatctt cgggctggcc cctggcaagg cccgggacag gaaggcctac
     4501 acggtcctcc tatacggaaa cggtccaggc tatgtgctca aggacggcgc ccggccggat
     4561 gttaccgaga gcgagagcgg gagccccgag tatcggcagc agtcagcagt gcccctggac
     4621 gaagagaccc acgcaggcga ggacgtggcg gtgttcgcgc gcggcccgca ggcgcacctg
     4681 gttcacggcg tgcaggagca gaccttcata gcgcacgtca tggccttcgc cgcctgcctg
     4741 gagccctaca ccgcctgcga cctggcgccc cccgccggca ccaccgacgc cgcgcacccg
     4801 ggtggcggag gactgaacga catcttcgag gctcagaaaa tcgaatggca cgaacatcac
     4861 catcaccatc accatcacca ttaaggccgg cctgtagtca ctggcacaac gcgttacccg
     4921 gtaagccaat cgggtataca cggtcgtcat actgcagaca gggttcttct actttgcaag
     4981 atagtcttga gtagtaaaat aaatagatag agaaaatcag cctcgactgt gccttctagt
     5041 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     5101 cccactgtcc tttcctaata aaatgaggaa attgcatcac aacactcaac cctatctcgg
     5161 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5221 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     5281 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     5341 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     5401 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     5461 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     5521 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     5581 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt
     5641 gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa
     5701 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     5761 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     5821 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     5881 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     5941 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     6001 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     6061 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     6121 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     6181 ctggagacat ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     6241 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     6301 tgacacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     6361 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     6421 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     6481 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     6541 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     6601 tggtcattac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6661 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6721 tggccccagc gctgcgatga taccgcgaga accacgctca ccggctccgg atttatcagc
     6781 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6841 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6901 gcgcaacgtt gttgccatcg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6961 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7021 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7081 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7141 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7201 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7261 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7321 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7381 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7441 ggcgacacgg aaatgttgaa tactcatatt cttcctt
//
