LOCUS       sequence-304011-        8653 bp    DNA     circular SYN 15-DEC-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 8653)
  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 8653)
  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-ALCAM-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8653
                     /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             4258..4293
                     /codon_start=1
                     /product="epitope tag derived from human Protein C (Stearns
                     et al., 1988)"
                     /label=Protein C tag
                     /translation="EDQVDPRLIDGK"
     CDS             4303..4326
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5986..6030
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6031..6057
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(6197..6214)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6203..6314
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6432..6452)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6437..6766
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6617..6752
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6679..6698
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6814..6861
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6880..7479
                     /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(6880..6899)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7376..7396
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7609..7730
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7646..7665)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7700..7719
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7783..8574,8575..8643))
                     /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     8406..8425
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8644..8653,1..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 cggccgcctg gtacacagtg
     2761 aatagcgcct acggcgacac catcatcatc ccttgcagac tggacgtgcc ccagaacctg
     2821 atgttcggca agtggaagta cgagaagccc gacggcagcc ccgtgttcat tgcctttaga
     2881 agcagcacca agaagtccgt ccagtacgac gacgtgcccg agtacaagga cagactgaac
     2941 ctgagcgaga actacaccct gagcatcagc aacgcccgga tcagcgacga gaagagattc
     3001 gtgtgcatgc tggtcaccga ggacaacgtg ttcgaggccc ctaccatcgt gaaggtgttc
     3061 aagcagccca gcaagcccga gatcgtgtct aaggccctgt tcctggaaac cgagcagctg
     3121 aagaagctgg gcgactgcat cagcgaggac agctaccccg atggcaacat cacctggtac
     3181 agaaacggca aggtgctgca ccctctggaa ggcgccgtgg tcatcatctt caagaaagaa
     3241 atggaccccg tgacgcagct gtacaccatg accagcacac tcgagtataa gaccaccaag
     3301 gccgacattc agatgccctt cacctgtagc gtgacctact acggccctag cggccagaaa
     3361 accatccact ctgagcaggc cgtgttcgac atctactacc ccaccgagca agtgaccatc
     3421 caggtgctgc ctccaaagaa cgccatcaaa gagggcgaca atatcaccct gaagtgcctc
     3481 ggcaacggca acccgcctcc tgaagagttc ctgttctacc tgcctggcca gcctgagggc
     3541 atcagaagct ccaacaccta cacactgacc gacgtgcgga gaaacgccac cggcgattac
     3601 aagtgcagcc tgatcgacaa aaagagcatg atcgccagca ccgccatcac cgtgcactac
     3661 ctggacctga gcctgaatcc tagcggcgaa gtgaccagac agatcggaga tgccctgcct
     3721 gtgtcctgta caatcagcgc cagcagaaat gccaccgtcg tgtggatgaa ggacaacatc
     3781 cggctgagaa gcagccccag ctttagcagc ctgcactatc aggacgccgg caactacgtg
     3841 tgcgagacag ccctgcaaga ggtggaaggc ctgaagaaga gagagtctct gaccctgatc
     3901 gtggaaggca agcctcagat caagatgacc aagaaaacag accccagcgg cctgagcaag
     3961 accatcatct gtcacgtgga aggattcccc aagcctgcca tccagtggac catcacaggc
     4021 agcggctccg tgatcaacca gaccgaggaa agcccctaca tcaacggccg gtactactcc
     4081 aagatcatca tcagccccga agagaacgtg accctgacct gcacagccga gaaccagctg
     4141 gaaagaaccg tgaacagcct gaacgtgtcc gccatcagca tccccgaaca cgatgaggcc
     4201 gacgagatct ccgacgagaa ccgcgagaaa gtgaacgacc aggccaaagg cgcgccggaa
     4261 gatcaagttg atccaagatt gattgatggc aagggcggat ctctggaggt gctgttccag
     4321 ggaccaggcg gaagcggcaa tcccggcggc gatctggccc cccagatgct gcgcgagctg
     4381 caggagacca atgccgccct gcaggacgtg cgcgagctgc tgcgccaccg cgtgaaggag
     4441 atcaccttcc tgaagaacac cgtgatggag tgcgacgcct gcggtatgca gccctctaga
     4501 ggctccggct ccatcatccc agttgaggag gagaacccgg acttctggaa ccgcgaggca
     4561 gccgaggccc tgggtgccgc caagaagctg cagcctgcac agacagccgc caagaacctc
     4621 atcatcttcc tgggcgatgg gatgggggtg tctacggtga cagctgccag gatcctaaaa
     4681 gggcagaaga aggacaaact ggggcctgag atacccctgg ccatggaccg cttcccatat
     4741 gtggctctgt ccaagacata caatgtagac aaacatgtgc cagacagtgg agccacagcc
     4801 acggcctacc tgtgcggggt caagggcaac ttccagacca ttggcttgag tgcagccgcc
     4861 cgctttaacc agtgcaacac gacacgcggc aacgaggtca tctccgtgat gaatcgggcc
     4921 aagaaagcag ggaagtcagt gggagtggta accaccacac gagtgcagca cgcctcgcca
     4981 gccggcacct acgcccacac ggtgaaccgc aactggtact cggacgccga cgtgcctgcc
     5041 tcggcccgcc aggaggggtg ccaggacatc gctacgcagc tcatctccaa catggacatt
     5101 gacgtgatcc taggtggagg ccgaaagtac atgtttcgca tgggaacccc agaccctgag
     5161 tacccagatg actacagcca aggtgggacc aggctggacg ggaagaatct ggtgcaggaa
     5221 tggctggcga agcgccaggg tgcccggtat gtgtggaacc gcactgagct catgcaggct
     5281 tccctggacc cgtctgtgac ccatctcatg ggtctctttg agcctggaga catgaaatac
     5341 gagatccacc gagactccac actggacccc tccctgatgg agatgacaga ggctgccctg
     5401 cgcctgctga gcaggaaccc ccgcggcttc ttcctcttcg tggagggtgg tcgcatcgac
     5461 catggtcatc atgaaagcag ggcttaccgg gcactgactg agacgatcat gttcgacgac
     5521 gccattgaga gggcgggcca gctcaccagc gaggaggaca cgctgagcct cgtcactgcc
     5581 gaccactccc acgtcttctc cttcggaggc taccccctgc gagggagctc catcttcggg
     5641 ctggcccctg gcaaggcccg ggacaggaag gcctacacgg tcctcctata cggaaacggt
     5701 ccaggctatg tgctcaagga cggcgcccgg ccggatgtta ccgagagcga gagcgggagc
     5761 cccgagtatc ggcagcagtc agcagtgccc ctggacgaag agacccacgc aggcgaggac
     5821 gtggcggtgt tcgcgcgcgg cccgcaggcg cacctggttc acggcgtgca ggagcagacc
     5881 ttcatagcgc acgtcatggc cttcgccgcc tgcctggagc cctacaccgc ctgcgacctg
     5941 gcgccccccg ccggcaccac cgacgccgcg cacccgggtg gcggaggact gaacgacatc
     6001 ttcgaggctc agaaaatcga atggcacgaa catcaccatc accatcacca tcaccattaa
     6061 ggccggcctg tagtcactgg cacaacgcgt tacccggtaa gccaatcggg tatacacggt
     6121 cgtcatactg cagacagggt tcttctactt tgcaagatag tcttgagtag taaaataaat
     6181 agatagagaa aatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     6241 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     6301 gaggaaattg catcacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     6361 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     6421 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     6481 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     6541 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     6601 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     6661 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     6721 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     6781 ttgtatatcc attttcggat ctgatcagca cgtgttgaca attaatcatc ggcatagtat
     6841 atcggcatag tataatacga caaggtgagg aactaaacca tgaccgagta caagcccacg
     6901 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     6961 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     7021 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     7081 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     7141 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     7201 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     7261 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     7321 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacatccgc gccccgcaac
     7381 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     7441 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac acgtgctacg agatttcgat
     7501 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     7561 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     7621 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     7681 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     7741 ataccgtcga cctctagcta gagcttggcg taatcatggt cattaccaat gcttaatcag
     7801 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     7861 cgtgtagata actacgatac gggagggctt accatctggc cccagcgctg cgatgatacc
     7921 gcgagaacca cgctcaccgg ctccggattt atcagcaata aaccagccag ccggaagggc
     7981 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     8041 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccatcgctac
     8101 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     8161 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     8221 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     8281 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     8341 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     8401 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     8461 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     8521 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     8581 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     8641 catattcttc ctt
//
