LOCUS       sequence-481689-        7963 bp    DNA     circular SYN 01-AUG-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 7963)
  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 7963)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 1, 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-CRTAM-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..7963
                     /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             3613..3636
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5296..5340
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5341..5367
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(5507..5524)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5513..5624
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5742..5762)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5747..6076
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5927..6062
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5989..6008
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6124..6171
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6190..6789
                     /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(6190..6209)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6686..6706
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6919..7040
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6956..6975)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7010..7029
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7093..7884,7885..7953))
                     /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     7716..7735
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7954..7963,1..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 cggccgccag cctgacaaat
     2761 cacaccgaga caatcaccgt ggaagagggc cagacactga ccctgaaatg cgtgaccagc
     2821 ctgcggaaga atagcagcct gcagtggctg acccctagcg gcttcaccat ctttctgaac
     2881 gagtaccccg ctctgaagaa cagcaagtac cagctgctgc accacagcgc caaccagctg
     2941 agcatcaccg tgcctaatgt gaccctgcag gacgagggcg tgtacaagtg tctgcactac
     3001 agcgacagcg tgtccaccaa agaagtgaaa gtcatcgtcc tggccacacc tttcaagccc
     3061 atcctggaag ccagcgtgat cagaaagcag aacggcgagg aacacgtggt gctgatgtgc
     3121 agcaccatga gaagcaagcc tccacctcag atcacctggc tgctgggcaa ttctatggaa
     3181 gtgtccggcg gcaccctgca cgagtttgag acagacggca agaagtgcaa caccaccagc
     3241 acactgatca tccacaccta cggcaaaaac agcaccgtgg actgcatcat ccggcacaga
     3301 ggactgcagg gaagaaagct ggtggcccct ttcagattcg aggacctggt caccgatgag
     3361 gaaacagcct ctgacgccct ggaacggaat agcctgtcta gtcaggaccc tcagcagcct
     3421 accagcacag tgtctgtgac cgaggatagc agcaccagcg agatcgacaa agaggaaaaa
     3481 gagcagacca cacaggaccc cgatctgacc acagaggcca atcctcagta tctgggcctc
     3541 gccagaaaga aaagcggagg cgcgccggaa gatcaagttg atccaagatt gattgatggc
     3601 aagggcggat ctctggaggt gctgttccag ggaccaggcg gaagcggcaa tcccggcggc
     3661 gatctggccc cccagatgct gcgcgagctg caggagacca atgccgccct gcaggacgtg
     3721 cgcgagctgc tgcgccaccg cgtgaaggag atcaccttcc tgaagaacac cgtgatggag
     3781 tgcgacgcct gcggtatgca gccctctaga ggctccggct ccatcatccc agttgaggag
     3841 gagaacccgg acttctggaa ccgcgaggca gccgaggccc tgggtgccgc caagaagctg
     3901 cagcctgcac agacagccgc caagaacctc atcatcttcc tgggcgatgg gatgggggtg
     3961 tctacggtga cagctgccag gatcctaaaa gggcagaaga aggacaaact ggggcctgag
     4021 atacccctgg ccatggaccg cttcccatat gtggctctgt ccaagacata caatgtagac
     4081 aaacatgtgc cagacagtgg agccacagcc acggcctacc tgtgcggggt caagggcaac
     4141 ttccagacca ttggcttgag tgcagccgcc cgctttaacc agtgcaacac gacacgcggc
     4201 aacgaggtca tctccgtgat gaatcgggcc aagaaagcag ggaagtcagt gggagtggta
     4261 accaccacac gagtgcagca cgcctcgcca gccggcacct acgcccacac ggtgaaccgc
     4321 aactggtact cggacgccga cgtgcctgcc tcggcccgcc aggaggggtg ccaggacatc
     4381 gctacgcagc tcatctccaa catggacatt gacgtgatcc taggtggagg ccgaaagtac
     4441 atgtttcgca tgggaacccc agaccctgag tacccagatg actacagcca aggtgggacc
     4501 aggctggacg ggaagaatct ggtgcaggaa tggctggcga agcgccaggg tgcccggtat
     4561 gtgtggaacc gcactgagct catgcaggct tccctggacc cgtctgtgac ccatctcatg
     4621 ggtctctttg agcctggaga catgaaatac gagatccacc gagactccac actggacccc
     4681 tccctgatgg agatgacaga ggctgccctg cgcctgctga gcaggaaccc ccgcggcttc
     4741 ttcctcttcg tggagggtgg tcgcatcgac catggtcatc atgaaagcag ggcttaccgg
     4801 gcactgactg agacgatcat gttcgacgac gccattgaga gggcgggcca gctcaccagc
     4861 gaggaggaca cgctgagcct cgtcactgcc gaccactccc acgtcttctc cttcggaggc
     4921 taccccctgc gagggagctc catcttcggg ctggcccctg gcaaggcccg ggacaggaag
     4981 gcctacacgg tcctcctata cggaaacggt ccaggctatg tgctcaagga cggcgcccgg
     5041 ccggatgtta ccgagagcga gagcgggagc cccgagtatc ggcagcagtc agcagtgccc
     5101 ctggacgaag agacccacgc aggcgaggac gtggcggtgt tcgcgcgcgg cccgcaggcg
     5161 cacctggttc acggcgtgca ggagcagacc ttcatagcgc acgtcatggc cttcgccgcc
     5221 tgcctggagc cctacaccgc ctgcgacctg gcgccccccg ccggcaccac cgacgccgcg
     5281 cacccgggtg gcggaggact gaacgacatc ttcgaggctc agaaaatcga atggcacgaa
     5341 catcaccatc accatcacca tcaccattaa ggccggcctg tagtcactgg cacaacgcgt
     5401 tacccggtaa gccaatcggg tatacacggt cgtcatactg cagacagggt tcttctactt
     5461 tgcaagatag tcttgagtag taaaataaat agatagagaa aatcagcctc gactgtgcct
     5521 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     5581 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcacaaca ctcaacccta
     5641 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5701 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     5761 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     5821 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     5881 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     5941 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     6001 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     6061 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca
     6121 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg
     6181 aactaaacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     6241 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     6301 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     6361 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     6421 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     6481 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     6541 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     6601 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     6661 gccttcctgg agacatccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     6721 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     6781 ggtgcctgac acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     6841 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     6901 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     6961 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     7021 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     7081 taatcatggt cattaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     7141 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     7201 accatctggc cccagcgctg cgatgatacc gcgagaacca cgctcaccgg ctccggattt
     7261 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7321 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7381 tagtttgcgc aacgttgttg ccatcgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7441 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7501 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7561 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7621 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7681 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7741 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7801 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7861 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7921 aataagggcg acacggaaat gttgaatact catattcttc ctt
//
