LOCUS       sequence-465769-        7362 bp    DNA     circular SYN 15-APR-2025
DEFINITION  expresses LaccID on the mammalian cell surface.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7362)
  TITLE     Directed evolution of LaccID for cell surface proximity labeling and
            EM
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7362)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pCMV_SS-HA-V5-LaccID-CD4 TM_pDisplay
FEATURES             Location/Qualifiers
     source          1..7362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        10..389
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..593
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     544..564
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     638..657
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        638..656
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     737..799
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTGD"
     CDS             800..826
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             854..895
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3113..3130)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3119..3343
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(3475..4063)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3555..3574)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4392..4439
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4464..4483
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4671..5465)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(4671..5465)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4782..4801)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5392..5411
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(5564..5583)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        5662..5853
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5810..5830
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(5913..6704,6705..6773))
                     /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     6536..6555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6774..6878)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6905..7360
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6992..7011)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7202..7223
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
       61 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      121 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      181 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      241 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      361 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      421 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      481 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      541 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc
      601 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga
      661 cccaagcttg gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattcgg
      721 cttggggata tccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt
      781 tccaggttcc actggtgact atccatatga tgttccagat tatgctgggg cccagccggc
      841 cagatctggc ggcggcaagc ccatccccaa ccccctgctg ggcctggaca gcaccgctag
      901 cgcgatagga cctgtggcag accttcacat cgccaatgca gacatcagcc cggatgggtt
      961 cactcgttcc gctgtgctag cgggtggaac atttcccggt cccttaataa cgggtaataa
     1021 aggagataat ttccggataa acgtcatcaa ccagcttacg gacacgacta tgttgaggtc
     1081 aaccagtata cattggcacg gattctttca acacgggaca agttgggctg acggccccgc
     1141 tttcgtcact cagtgcccca tcgcaccggg taacagtttt ctttacaact tcactgtccc
     1201 ggatcaagcg ggcacgttct ggtatcatag tcacctaagt acccagtatt gtgacggact
     1261 tagtggtccg tttgtagttt acgaccccaa cgatccccat cgtagcttat acgatgtgga
     1321 tgatgaaagc actgtaatta ctcttgcgga ctggtaccac accccggctc cccagttggc
     1381 cgctgcgccg cccgtcccag atagcaccct aataaacggg aaaggaaggt atgccggagg
     1441 cccaacgtcc ccgttagccg tgataaacgt agagcaggga aaaagatata ggtttcgtct
     1501 tatatctatt tcctgtgatc cgaattttac atttagcatt gatggccata atatgactat
     1561 cattgaagct gatggggtga acacgcaacc actgaccgtg gactctatcc agattttcgc
     1621 tggccaaaga tattccttcg tcctgaatgc gaaccagcct gttggtaact attggataag
     1681 agcgaatcca aatataggaa cgacaggatt cgcaggtggt attaatagtg cgatcctacg
     1741 ttatgtagga gctccagaag cagaccctac aacgacccaa acaacatcca caaatccagt
     1801 gaacgaaaca aacctacacc ccttggagaa ccccggagcc ccggggaatc cgactccggg
     1861 tggtgctgat gtgaacatca accttaacat cggattctcc tcaacgggac aattcaccat
     1921 aaacggtgtt tccttcatac cacccaccgt tcccgtcttg ttgcaaatcc tgtccggggc
     1981 gcgtacggca caagatctgc ttcctagcgg ctctgtttat acattaccgc cgaacaaagt
     2041 catagaaatt agtatgccag gcggcgctag cgcagccggg gggcctcacc cgttccattt
     2101 gcacggccac acttttgatg tggtacgttc agcgggatca tcaacttaca actacgtgaa
     2161 tccagtcaga agagatgtcg tctccattgg aacggcggga gataacgtga caattaggtt
     2221 cactacggac aacccgggtc catggttcct acattgtcat atcgactggc atcttgaggc
     2281 cggtttagcc gtcgtattcg ctgaagacac acctgatatc gcaagcgcta atcccgttcc
     2341 ggctgcgtgg gacgacctat gccctattta tgatgcgtta agccccgacg atcaaccgcg
     2401 gctgcaggtc gacctcgact tccagaaggc ctccagcata gtctataaga aagaggggga
     2461 acaggtggag ttctccttcc cactcgcctt tacagttgaa aagctgacgg gcagtggcga
     2521 gctgtggtgg caggcggaga gggcttcctc ctccaagtct tggatcacct ttgacctgaa
     2581 gaacaaggaa gtgtctgtaa aacgggttac ccaggaccct aagctccaga tgggcaagaa
     2641 gctcccgctc cacctcaccc tgccccaggc cttgcctcag tatgctggct ctggaaacct
     2701 caccctggcc cttgaagcga aaacaggaaa gttgcatcag gaagtgaacc tggtggtgat
     2761 gagagccact cagctccaga aaaatttgac ctgtgaggtg tggggaccca cctcccctaa
     2821 gctgatgctg agcttgaaac tggagaacaa ggaggcaaag gtctcgaagc gggagaaggc
     2881 ggtgtgggtg ctgaaccctg aggcggggat gtggcagtgt ctgctgagtg actcgggaca
     2941 ggtcctgctg gaatccaaca tcaaggttct gcccacatgg tccaccccgg tgcagccaat
     3001 ggccctgatt gtgctggggg gcgtcgccgg cctcctgctt ttcattgggc taggcatctt
     3061 cttctgtgtc aggtgccggc accgaaggcg ctaggcggcc gctcgagatc agcctcgact
     3121 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     3181 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     3241 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     3301 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     3361 accagtggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3421 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3481 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3541 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3601 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3661 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3721 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3781 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3841 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3901 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3961 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4021 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4081 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4141 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4201 ctaaagtata tatgagtaac ctgaggctat ggcagggcct gccgccccga cgttggctgc
     4261 gagccctggg ccttcacccg aacttggggg gtggggtggg gaaaaggaag aaacgcgggc
     4321 gtattggccc caatggggtc tcggtggggt atcgacagag tgccagccct gggaccgaac
     4381 cccgcgttta tgaacaaacg acccaacacc gtgcgtttta ttctgtcttt ttattgccgt
     4441 catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tccccctagg
     4501 gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg
     4561 gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga
     4621 tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac
     4681 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc
     4741 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac
     4801 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag
     4861 cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc
     4921 tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga
     4981 tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc
     5041 tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc
     5101 cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg
     5161 agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg
     5221 tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg
     5281 tcttgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc
     5341 tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca
     5401 tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca
     5461 atcatgcgaa acgatcctca tcctgtctct tgatcgatct ttgcaaaagc ctaggcctcc
     5521 aaaaaagcct cctcactact tctggaatag ctcagaggcc gggcctcggc ctctgcataa
     5581 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     5641 gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc
     5701 atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga
     5761 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga
     5821 cacacattcc acagctggtt ctttccgcct caggactctt cctttttcaa taaatcaatc
     5881 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5941 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6001 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6061 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6121 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6181 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6241 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6301 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6361 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6421 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6481 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6541 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6601 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6661 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6721 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6781 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6841 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6901 cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     6961 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     7021 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     7081 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     7141 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     7201 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     7261 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     7321 tttaacgcga attttaacaa aatattaacg cttacaattt ac
//
