LOCUS       sequence-302927-        7714 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression of cell-surface protein extracellular domain 
            fused to Fc(DAPA)-Avi-6xHis. Protein is secreted from cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7714)
  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 7714)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-MCAM-Fc(DAPA)-AviTag-6xHis
FEATURES             Location/Qualifiers
     source          1..7714
                     /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             5056..5100
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             5101..5118
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5258..5275)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5264..5375
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5493..5513)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5498..5827
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5678..5813
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        5875..5922
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5941..6540
                     /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(5941..5960)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6437..6457
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6670..6791
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6707..6726)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6761..6780
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6844..7635,7636..7704))
                     /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     7467..7486
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7705..7714,1..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 cgccgggcgg cgacaagacc
     4381 cacacctgcc ccccctgccc agctccagaa ctgctgggag ggccttccgt gttcctgttc
     4441 ccccccaagc ccaaggacac cctgatgatc agcaggaccc ccgaggtgac ctgcgtggtg
     4501 gtggccgtgt cccacgagga cccagaggtg aagttcaact ggtacgtgga cggcgtggag
     4561 gtgcacaacg ccaagaccaa gcccagagag gagcagtaca acagcaccta cagggtggtg
     4621 tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aagaatacaa gtgcaaagtc
     4681 tccaacaagg ccctggctgc cccaatcgaa aagacaatca gcaaggccaa gggccagcca
     4741 cgggagcccc aggtgtacac cctgcccccc agccgggagg agatgaccaa gaaccaggtg
     4801 tccctgacct gtctggtgaa gggcttctac cccagcgata tcgccgtgga gtgggagagc
     4861 aacggccagc ccgagaacaa ctacaagacc acccccccag tgctggacag cgacggcagc
     4921 ttcttcctgt acagcaagct gaccgtggac aagtccaggt ggcagcaggg caacgtgttc
     4981 agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg
     5041 agccccggcg gcagcggtct taatgatatt ttcgaggctc agaagattga gtggcatgaa
     5101 caccaccacc atcaccacta aggccggcct gtagtcactg gcacaacgcg ttacccggta
     5161 agccaatcgg gtatacacgg tcgtcatact gcagacaggg ttcttctact ttgcaagata
     5221 gtcttgagta gtaaaataaa tagatagaga aaatcagcct cgactgtgcc ttctagttgc
     5281 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     5341 actgtccttt cctaataaaa tgaggaaatt gcatcacaac actcaaccct atctcggtct
     5401 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5461 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     5521 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     5581 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     5641 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     5701 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     5761 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     5821 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac
     5881 aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc
     5941 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     6001 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     6061 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     6121 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     6181 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     6241 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     6301 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     6361 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     6421 gagacatccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     6481 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     6541 cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     6601 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     6661 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     6721 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     6781 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     6841 tcattaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6901 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6961 ccccagcgct gcgatgatac cgcgagaacc acgctcaccg gctccggatt tatcagcaat
     7021 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7081 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7141 caacgttgtt gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7201 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7261 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7321 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7381 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7441 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7501 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7561 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7621 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7681 gacacggaaa tgttgaatac tcatattctt cctt
//
