LOCUS       sequence-488901-        8761 bp    DNA     circular SYN 09-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 8761)
  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 8761)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 9, 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-MCAM-COMP5AP-AviTag-9xHis
FEATURES             Location/Qualifiers
     source          1..8761
                     /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             4411..4434
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6094..6138
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             6139..6165
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     primer_bind     complement(6305..6322)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6311..6422
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6540..6560)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6545..6874
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6725..6860
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        6922..6969
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6988..7587
                     /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(6988..7007)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7484..7504
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7717..7838
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7754..7773)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7808..7827
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(7891..8682,8683..8751))
                     /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     8514..8533
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8752..8761,1..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 cggccgccgt tccaggcgaa
     2761 gctgaacaac ctgctcctga gctggttgag gtggaagtgg gatctacagc cctgctgaag
     2821 tgtggcctga gccagtctca gggcaatctg agccacgtgg actggttcag cgtgcacaaa
     2881 gagaagcgga ccctgatctt cagagtgcgg caaggacagg gacagagcga gcctggcgaa
     2941 tacgagcaaa gactgagcct gcaggataga ggcgccacac tggctctgac acaagtgacc
     3001 cctcaggacg agcggatctt cctgtgccaa ggcaagaggc ccagaagcca agagtacaga
     3061 atccagctgc gggtgtacaa ggcccctgag gaacccaaca tccaagtgaa ccctctgggc
     3121 atccccgtga acagcaaaga acctgaagag gtggccacat gcgtgggcag aaacggctac
     3181 cccattcctc aagtgatctg gtacaagaac ggcaggcctc tgaaagagga aaagaaccgg
     3241 gtccacatcc agagcagcca gacagtggaa agcagcggcc tgtacacact gcagagcatt
     3301 ctgaaggccc agctggtcaa agaggacaag gacgcccagt tctactgcga gctgaactac
     3361 agactgccca gcggcaacca catgaaggaa tccagagaag tgaccgtgcc tgtgttctac
     3421 cccaccgaga aagtgtggct ggaagtggaa cccgtgggca tgctgaaaga aggcgacaga
     3481 gtcgagatca gatgcctggc cgatggcaac cctcctccac acttttccat cagcaagcag
     3541 aaccccagca ccagagaggc cgaggaagaa accaccaacg acaatggcgt gctggtgctc
     3601 gagcctgcca gaaaagaaca cagcggcaga tacgagtgcc agggcctcga tctggatacc
     3661 atgatcagcc tgctgagcga gccacaagag ctgctggtca actatgtgtc cgatgtgcga
     3721 gtgtctcccg ccgctcctga gagacaagag ggaagctctc tgacactgac atgcgaggcc
     3781 gagtccagcc aggacctgga atttcagtgg ctgagagaag agacaggcca ggtgctggaa
     3841 agaggccctg ttctgcagct ccacgatctg aagagagaag ccggcggagg ctacagatgt
     3901 gtggcctctg ttcctagcat ccccggcctg aatagaaccc agctcgtgaa cgtggccatc
     3961 ttcggacctc catggatggc cttcaaagag cggaaagtct gggtcaaaga aaacatggtg
     4021 ctgaacctga gctgcgaggc ctctggacac cctagaccta ccatcagctg gaatgtgaat
     4081 ggcaccgcca gcgagcagga tcaggaccct caaagagtgc tgagcaccct gaatgtgctg
     4141 gtcacccctg aactgctgga aaccggcgtg gaatgtaccg ccagcaacga cctgggcaag
     4201 aacaccagca tcctgtttct ggaactggtc aacctgacca cactgacccc tgacagcaat
     4261 accaccacag gcctgagcac aagcaccgcc tctccacaca caagagccaa tagcacctcc
     4321 accgagagaa agctgcctga gcctgaaagc cgcggaggcg cgccggaaga tcaagttgat
     4381 ccaagattga ttgatggcaa gggcggatct ctggaggtgc tgttccaggg accaggcgga
     4441 agcggcaatc ccggcggcga tctggccccc cagatgctgc gcgagctgca ggagaccaat
     4501 gccgccctgc aggacgtgcg cgagctgctg cgccaccgcg tgaaggagat caccttcctg
     4561 aagaacaccg tgatggagtg cgacgcctgc ggtatgcagc cctctagagg ctccggctcc
     4621 atcatcccag ttgaggagga gaacccggac ttctggaacc gcgaggcagc cgaggccctg
     4681 ggtgccgcca agaagctgca gcctgcacag acagccgcca agaacctcat catcttcctg
     4741 ggcgatggga tgggggtgtc tacggtgaca gctgccagga tcctaaaagg gcagaagaag
     4801 gacaaactgg ggcctgagat acccctggcc atggaccgct tcccatatgt ggctctgtcc
     4861 aagacataca atgtagacaa acatgtgcca gacagtggag ccacagccac ggcctacctg
     4921 tgcggggtca agggcaactt ccagaccatt ggcttgagtg cagccgcccg ctttaaccag
     4981 tgcaacacga cacgcggcaa cgaggtcatc tccgtgatga atcgggccaa gaaagcaggg
     5041 aagtcagtgg gagtggtaac caccacacga gtgcagcacg cctcgccagc cggcacctac
     5101 gcccacacgg tgaaccgcaa ctggtactcg gacgccgacg tgcctgcctc ggcccgccag
     5161 gaggggtgcc aggacatcgc tacgcagctc atctccaaca tggacattga cgtgatccta
     5221 ggtggaggcc gaaagtacat gtttcgcatg ggaaccccag accctgagta cccagatgac
     5281 tacagccaag gtgggaccag gctggacggg aagaatctgg tgcaggaatg gctggcgaag
     5341 cgccagggtg cccggtatgt gtggaaccgc actgagctca tgcaggcttc cctggacccg
     5401 tctgtgaccc atctcatggg tctctttgag cctggagaca tgaaatacga gatccaccga
     5461 gactccacac tggacccctc cctgatggag atgacagagg ctgccctgcg cctgctgagc
     5521 aggaaccccc gcggcttctt cctcttcgtg gagggtggtc gcatcgacca tggtcatcat
     5581 gaaagcaggg cttaccgggc actgactgag acgatcatgt tcgacgacgc cattgagagg
     5641 gcgggccagc tcaccagcga ggaggacacg ctgagcctcg tcactgccga ccactcccac
     5701 gtcttctcct tcggaggcta ccccctgcga gggagctcca tcttcgggct ggcccctggc
     5761 aaggcccggg acaggaaggc ctacacggtc ctcctatacg gaaacggtcc aggctatgtg
     5821 ctcaaggacg gcgcccggcc ggatgttacc gagagcgaga gcgggagccc cgagtatcgg
     5881 cagcagtcag cagtgcccct ggacgaagag acccacgcag gcgaggacgt ggcggtgttc
     5941 gcgcgcggcc cgcaggcgca cctggttcac ggcgtgcagg agcagacctt catagcgcac
     6001 gtcatggcct tcgccgcctg cctggagccc tacaccgcct gcgacctggc gccccccgcc
     6061 ggcaccaccg acgccgcgca cccgggtggc ggaggactga acgacatctt cgaggctcag
     6121 aaaatcgaat ggcacgaaca tcaccatcac catcaccatc accattaagg ccggcctgta
     6181 gtcactggca caacgcgtta cccggtaagc caatcgggta tacacggtcg tcatactgca
     6241 gacagggttc ttctactttg caagatagtc ttgagtagta aaataaatag atagagaaaa
     6301 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     6361 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     6421 tcacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     6481 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     6541 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     6601 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     6661 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     6721 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     6781 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     6841 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     6901 tttcggatct gatcagcacg tgttgacaat taatcatcgg catagtatat cggcatagta
     6961 taatacgaca aggtgaggaa ctaaaccatg accgagtaca agcccacggt gcgcctcgcc
     7021 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     7081 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     7141 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     7201 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     7261 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     7321 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     7381 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     7441 gagcgcgccg gggtgcccgc cttcctggag acatccgcgc cccgcaacct ccccttctac
     7501 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     7561 tgcatgaccc gcaagcccgg tgcctgacac gtgctacgag atttcgattc caccgccgcc
     7621 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     7681 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     7741 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     7801 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     7861 tctagctaga gcttggcgta atcatggtca ttaccaatgc ttaatcagtg aggcacctat
     7921 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     7981 tacgatacgg gagggcttac catctggccc cagcgctgcg atgataccgc gagaaccacg
     8041 ctcaccggct ccggatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     8101 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     8161 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc atcgctacag gcatcgtggt
     8221 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     8281 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     8341 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     8401 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     8461 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     8521 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     8581 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     8641 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     8701 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tattcttcct
     8761 t
//
