LOCUS       sequence-464944-        8473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of secreted protein fused to 
            COMP5AP-AviTag-9xHis..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8473)
  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 8473)
  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-ROBO4-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8473
                     /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             4078..4113
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             4123..4146
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5806..5850
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5851..5877
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(6017..6034)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6023..6134
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6252..6272)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6257..6586
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6437..6572
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6499..6518
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6634..6681
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6700..7299
                     /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(6700..6719)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7196..7216
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7429..7550
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7466..7485)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7520..7539
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7603..8394,8395..8463))
                     /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     8226..8245
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8464..8473,1..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 agattctcca
     2761 cctcagattc tggtgcaccc tcaggaccag ctgtttcaag gacctggacc tgccagaatg
     2821 agctgtcagg cttctggaca gcctcctcca accatcagat ggctgctgaa tggccagcct
     2881 ctgagcatgg tgcctccaga tcctcaccat ctgctgcctg atggaacact gctgctgctt
     2941 cagcctcctg ccagaggaca tgctcatgat ggacaggccc tgagcaccga tctgggcgtg
     3001 tacacatgcg aggccagcaa tagactgggc acagctgtta gcagaggcgc cagactgtct
     3061 gtggccgtgc tgagagagga tttccagatc cagcctagag acatggtggc cgtcgtgggc
     3121 gagcagttta cactggaatg tggacctcca tggggacacc ctgagcctac agtgtcttgg
     3181 tggaaggatg gcaagccact ggctctgcaa cctggcagac ataccgtttc tggcggcagc
     3241 ctgctgatgg ctagagccga gaaatccgac gagggcacct atatgtgcgt ggccacaaat
     3301 agcgccggac acagagaatc cagagccgct agagtgtcca tccaagagcc tcaggattac
     3361 accgagcctg tggaactgct ggccgtgcgg attcagctgg aaaacgtgac cctgctgaac
     3421 cccgatcctg ccgagggacc taaacctaga cctgctgtgt ggctgagctg gaaggtttcc
     3481 ggacctgctg ctcctgctca gagctacaca gccctgttca gaacacagac agctccaggt
     3541 ggacaaggcg ccccttgggc tgaagaactt ctggctggat ggcagtctgc cgaactcgga
     3601 ggactgcact ggggccaaga ctacgagttt aaagtgcggc ctagcagcgg cagagctaga
     3661 ggccctgatt ctaacgtcct gctgctgaga ctgcccgaga aagtgcctag cgctcctcca
     3721 caagaagtga ccctgaagcc tggcaatggc accgtgtttg tgtcttgggt gccacctcca
     3781 gccgagaacc acaatggcat catccggggc tatcaagtgt ggtccctggg caatacttcc
     3841 ctgcctcctg ctaattggac cgttgtggga gagcagaccc agctcgagat tgccacacac
     3901 atgcctggca gctattgtgt gcaggttgcc gctgttacag gtgctggtgc tggcgaacct
     3961 agcagacctg tttgtctgct gctggaacag gccatggaaa gagccacaca agagcctagc
     4021 gagcacggcc cttggacact ggaacaactg agggccacac tgaagcgagg cgcgccggaa
     4081 gatcaagttg atccaagatt gattgatggc aagggcggat ctctggaggt gctgttccag
     4141 ggaccaggcg gaagcggcaa tcccggcggc gatctggccc cccagatgct gcgcgagctg
     4201 caggagacca atgccgccct gcaggacgtg cgcgagctgc tgcgccaccg cgtgaaggag
     4261 atcaccttcc tgaagaacac cgtgatggag tgcgacgcct gcggtatgca gccctctaga
     4321 ggctccggct ccatcatccc agttgaggag gagaacccgg acttctggaa ccgcgaggca
     4381 gccgaggccc tgggtgccgc caagaagctg cagcctgcac agacagccgc caagaacctc
     4441 atcatcttcc tgggcgatgg gatgggggtg tctacggtga cagctgccag gatcctaaaa
     4501 gggcagaaga aggacaaact ggggcctgag atacccctgg ccatggaccg cttcccatat
     4561 gtggctctgt ccaagacata caatgtagac aaacatgtgc cagacagtgg agccacagcc
     4621 acggcctacc tgtgcggggt caagggcaac ttccagacca ttggcttgag tgcagccgcc
     4681 cgctttaacc agtgcaacac gacacgcggc aacgaggtca tctccgtgat gaatcgggcc
     4741 aagaaagcag ggaagtcagt gggagtggta accaccacac gagtgcagca cgcctcgcca
     4801 gccggcacct acgcccacac ggtgaaccgc aactggtact cggacgccga cgtgcctgcc
     4861 tcggcccgcc aggaggggtg ccaggacatc gctacgcagc tcatctccaa catggacatt
     4921 gacgtgatcc taggtggagg ccgaaagtac atgtttcgca tgggaacccc agaccctgag
     4981 tacccagatg actacagcca aggtgggacc aggctggacg ggaagaatct ggtgcaggaa
     5041 tggctggcga agcgccaggg tgcccggtat gtgtggaacc gcactgagct catgcaggct
     5101 tccctggacc cgtctgtgac ccatctcatg ggtctctttg agcctggaga catgaaatac
     5161 gagatccacc gagactccac actggacccc tccctgatgg agatgacaga ggctgccctg
     5221 cgcctgctga gcaggaaccc ccgcggcttc ttcctcttcg tggagggtgg tcgcatcgac
     5281 catggtcatc atgaaagcag ggcttaccgg gcactgactg agacgatcat gttcgacgac
     5341 gccattgaga gggcgggcca gctcaccagc gaggaggaca cgctgagcct cgtcactgcc
     5401 gaccactccc acgtcttctc cttcggaggc taccccctgc gagggagctc catcttcggg
     5461 ctggcccctg gcaaggcccg ggacaggaag gcctacacgg tcctcctata cggaaacggt
     5521 ccaggctatg tgctcaagga cggcgcccgg ccggatgtta ccgagagcga gagcgggagc
     5581 cccgagtatc ggcagcagtc agcagtgccc ctggacgaag agacccacgc aggcgaggac
     5641 gtggcggtgt tcgcgcgcgg cccgcaggcg cacctggttc acggcgtgca ggagcagacc
     5701 ttcatagcgc acgtcatggc cttcgccgcc tgcctggagc cctacaccgc ctgcgacctg
     5761 gcgccccccg ccggcaccac cgacgccgcg cacccgggtg gcggaggact gaacgacatc
     5821 ttcgaggctc agaaaatcga atggcacgaa catcaccatc accatcacca tcaccattaa
     5881 ggccggcctg tagtcactgg cacaacgcgt tacccggtaa gccaatcggg tatacacggt
     5941 cgtcatactg cagacagggt tcttctactt tgcaagatag tcttgagtag taaaataaat
     6001 agatagagaa aatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     6061 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     6121 gaggaaattg catcacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     6181 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     6241 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     6301 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     6361 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     6421 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     6481 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     6541 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     6601 ttgtatatcc attttcggat ctgatcagca cgtgttgaca attaatcatc ggcatagtat
     6661 atcggcatag tataatacga caaggtgagg aactaaacca tgaccgagta caagcccacg
     6721 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     6781 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     6841 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     6901 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     6961 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     7021 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     7081 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     7141 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacatccgc gccccgcaac
     7201 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     7261 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac acgtgctacg agatttcgat
     7321 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     7381 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     7441 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     7501 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     7561 ataccgtcga cctctagcta gagcttggcg taatcatggt cattaccaat gcttaatcag
     7621 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     7681 cgtgtagata actacgatac gggagggctt accatctggc cccagcgctg cgatgatacc
     7741 gcgagaacca cgctcaccgg ctccggattt atcagcaata aaccagccag ccggaagggc
     7801 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     7861 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccatcgctac
     7921 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     7981 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     8041 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     8101 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     8161 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     8221 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     8281 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     8341 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     8401 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     8461 catattcttc ctt
//
