LOCUS       sequence-491299-        8335 bp    DNA     circular SYN 31-OCT-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 8335)
  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 8335)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 31, 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-CEACAM1-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8335
                     /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             3985..4008
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5668..5712
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5713..5739
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(5879..5896)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5885..5996
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6114..6134)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6119..6448
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6299..6434
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6361..6380
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6496..6543
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6562..7161
                     /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(6562..6581)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7058..7078
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7291..7412
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7328..7347)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7382..7401
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7465..8256,8257..8325))
                     /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     8088..8107
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8326..8335,1..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 actgacaaca
     2761 gagtccatgc ctttcaacgt ggccgagggc aaagaggtgc tgctgctggt tcacaacctg
     2821 cctcagcagc tgttcggcta cagctggtac aagggcgaga gagtggacgg caacagacag
     2881 atcgtgggct acgccatcgg aacccagcaa gctacacctg gacctgccaa cagcggcaga
     2941 gagacaatct accctaacgc cagcctgctg atccagaacg tgacccagaa cgacaccggc
     3001 ttctacaccc tgcaagtgat caagagcgac ctggtcaacg aggaagccac cggccagttt
     3061 cacgtgtacc ctgagctgcc caagcctagc atcagcagca acaacagcaa ccccgtggaa
     3121 gataaggacg ccgtggcctt tacctgcgag cccgagacac aggataccac ctacctgtgg
     3181 tggatcaaca accagagcct gccagtgtct cccagactgc agctgagcaa cggcaatcgg
     3241 acactgacac tgctgagcgt gaccagaaac gatacaggcc cctacgagtg cgagattcag
     3301 aaccccgtgt ccgccaatag aagcgacccc gtgacactga acgtgacata cggccccgac
     3361 acacccacaa tcagccccag cgatacctac tacagacctg gcgccaatct gagcctgtcc
     3421 tgctacgccg cttccaatcc tcctgctcag tacagctggc tgatcaatgg cacctttcag
     3481 cagagcaccc aagagctgtt catccccaac atcaccgtga acaactccgg cagctacaca
     3541 tgccacgcca acaatagcgt gaccggctgc aaccggacca ccgtgaaaac catcatcgtg
     3601 accgagctga gccccgtggt ggccaagcct cagattaagg ccagcaagac cacagtgacc
     3661 ggcgacaagg atagcgtgaa cctgacctgc agcaccaacg ataccggcat cagcatccgg
     3721 tggttcttca agaatcagtc cctgcctagc tccgagcgga tgaagctgtc tcagggcaac
     3781 accacactga gcatcaaccc tgtgaaaaga gaggacgccg gcacctattg gtgcgaggtg
     3841 ttcaacccca tcagcaagaa ccagagcgac cccatcatgc tgaatgtgaa ctacaacgcc
     3901 ctgcctcaag agaacggact gtctcccggt ggcgcgccgg aagatcaagt tgatccaaga
     3961 ttgattgatg gcaagggcgg atctctggag gtgctgttcc agggaccagg cggaagcggc
     4021 aatcccggcg gcgatctggc cccccagatg ctgcgcgagc tgcaggagac caatgccgcc
     4081 ctgcaggacg tgcgcgagct gctgcgccac cgcgtgaagg agatcacctt cctgaagaac
     4141 accgtgatgg agtgcgacgc ctgcggtatg cagccctcta gaggctccgg ctccatcatc
     4201 ccagttgagg aggagaaccc ggacttctgg aaccgcgagg cagccgaggc cctgggtgcc
     4261 gccaagaagc tgcagcctgc acagacagcc gccaagaacc tcatcatctt cctgggcgat
     4321 gggatggggg tgtctacggt gacagctgcc aggatcctaa aagggcagaa gaaggacaaa
     4381 ctggggcctg agatacccct ggccatggac cgcttcccat atgtggctct gtccaagaca
     4441 tacaatgtag acaaacatgt gccagacagt ggagccacag ccacggccta cctgtgcggg
     4501 gtcaagggca acttccagac cattggcttg agtgcagccg cccgctttaa ccagtgcaac
     4561 acgacacgcg gcaacgaggt catctccgtg atgaatcggg ccaagaaagc agggaagtca
     4621 gtgggagtgg taaccaccac acgagtgcag cacgcctcgc cagccggcac ctacgcccac
     4681 acggtgaacc gcaactggta ctcggacgcc gacgtgcctg cctcggcccg ccaggagggg
     4741 tgccaggaca tcgctacgca gctcatctcc aacatggaca ttgacgtgat cctaggtgga
     4801 ggccgaaagt acatgtttcg catgggaacc ccagaccctg agtacccaga tgactacagc
     4861 caaggtggga ccaggctgga cgggaagaat ctggtgcagg aatggctggc gaagcgccag
     4921 ggtgcccggt atgtgtggaa ccgcactgag ctcatgcagg cttccctgga cccgtctgtg
     4981 acccatctca tgggtctctt tgagcctgga gacatgaaat acgagatcca ccgagactcc
     5041 acactggacc cctccctgat ggagatgaca gaggctgccc tgcgcctgct gagcaggaac
     5101 ccccgcggct tcttcctctt cgtggagggt ggtcgcatcg accatggtca tcatgaaagc
     5161 agggcttacc gggcactgac tgagacgatc atgttcgacg acgccattga gagggcgggc
     5221 cagctcacca gcgaggagga cacgctgagc ctcgtcactg ccgaccactc ccacgtcttc
     5281 tccttcggag gctaccccct gcgagggagc tccatcttcg ggctggcccc tggcaaggcc
     5341 cgggacagga aggcctacac ggtcctccta tacggaaacg gtccaggcta tgtgctcaag
     5401 gacggcgccc ggccggatgt taccgagagc gagagcggga gccccgagta tcggcagcag
     5461 tcagcagtgc ccctggacga agagacccac gcaggcgagg acgtggcggt gttcgcgcgc
     5521 ggcccgcagg cgcacctggt tcacggcgtg caggagcaga ccttcatagc gcacgtcatg
     5581 gccttcgccg cctgcctgga gccctacacc gcctgcgacc tggcgccccc cgccggcacc
     5641 accgacgccg cgcacccggg tggcggagga ctgaacgaca tcttcgaggc tcagaaaatc
     5701 gaatggcacg aacatcacca tcaccatcac catcaccatt aaggccggcc tgtagtcact
     5761 ggcacaacgc gttacccggt aagccaatcg ggtatacacg gtcgtcatac tgcagacagg
     5821 gttcttctac tttgcaagat agtcttgagt agtaaaataa atagatagag aaaatcagcc
     5881 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     5941 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcacaa
     6001 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     6061 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     6121 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     6181 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     6241 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     6301 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     6361 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     6421 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     6481 atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac
     6541 gacaaggtga ggaactaaac catgaccgag tacaagccca cggtgcgcct cgccacccgc
     6601 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     6661 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     6721 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     6781 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     6841 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     6901 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     6961 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     7021 gccggggtgc ccgccttcct ggagacatcc gcgccccgca acctcccctt ctacgagcgg
     7081 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     7141 acccgcaagc ccggtgcctg acacgtgcta cgagatttcg attccaccgc cgccttctat
     7201 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     7261 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     7321 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     7381 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     7441 tagagcttgg cgtaatcatg gtcattacca atgcttaatc agtgaggcac ctatctcagc
     7501 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7561 acgggagggc ttaccatctg gccccagcgc tgcgatgata ccgcgagaac cacgctcacc
     7621 ggctccggat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     7681 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     7741 ttcgccagtt aatagtttgc gcaacgttgt tgccatcgct acaggcatcg tggtgtcacg
     7801 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7861 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7921 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7981 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     8041 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     8101 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     8161 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     8221 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     8281 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatattct tcctt
//
