LOCUS       sequence-425593-        7365 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of transferrin receptor transmembrane domain and 
            photocaged SpyCatcher003(K31HCK)-sfGFP on mammalian cell surface..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7365)
  AUTHORS   Rahikainen R, Vester SK, Turkki P, Janosko CP, Deiters A, Hytonen 
            VP, Howarth M
  TITLE     Visible Light-Induced Specific Protein Reaction Delineates Early 
            Stages of Cell Adhesion.
  JOURNAL   J Am Chem Soc. 2023 Nov 15;145(45):24459-24465. doi: 
            10.1021/jacs.3c07827. Epub 2023 Oct 31.
  PUBMED    38104267
REFERENCE   2  (bases 1 to 7365)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/jacs.3c07827"; journalName: "J Am Chem Soc"; date: 
            "2023-11-15- 15"; volume: "145"; issue: "45"; pages: "24459-24465"
COMMENT     Sequence Label: TfR-SpyCatcher003(K31TAG)-sfGFP-MycTag-CTag
FEATURES             Location/Qualifiers
     source          1..7365
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(105..319)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(165..184)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     promoter        complement(430..670)
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     primer_bind     complement(480..499)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(650..670)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(678..689)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     enhancer        760..1139
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1140..1343
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1294..1314
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1388..1407
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1388..1406
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2087..2800
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2801..2830
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     promoter        complement(2918..2936)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2919..2936)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2956..2973)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2962..3186
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3232..3660
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3319..3338)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3529..3550
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3669..3689)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3674..4003
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3854..3989
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3916..3935
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4070..4864
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4124..4143)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4734..4753
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5038..5159
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5075..5094)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5129..5148
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5208..5224)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5208..5224)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5221..5243)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5232..5248
                     /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)."
     promoter        complement(join(5256..5262,5263..5280,5281..5286))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5301..5322
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5439..5456)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5610..6198)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5690..5709)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6369..7160,7161..7229))
                     /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     6992..7011
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7230..7334)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctaga tccaaaaaac ggaaaccccg ggaatctaac ccggctgaac
       61 ggatttagag tccgttcgat ctacatgatc aggttcccag agctgtggtc tcatacagaa
      121 cttataagat tcccaaatcc aaagacattt cacgtttatg gtgatttccc agaacacata
      181 gcgacatgca aatattgcag ggcgccactc ccctgtccct cacagccatc ttcctgccag
      241 ggcgcacgcg cgctgggtgt tcccgcctag tgacactggg cccgcgattc cttggagcgg
      301 gttgatgacg tcagcgttcg aattgactag caaaaaccgc acttgtcaga tccaaaaaac
      361 ggggagtacg ggagtttcac ccgcctgcac gaatttagag tccgtgtgat ctattcgatc
      421 aacccccccg tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta
      481 cggtaagcat atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa
      541 attattactt tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa
      601 ttctaattat ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa
      661 ataggccctc ttcctgcccg accttcaatt gcatgaagaa tctgcttagg gttaggcgtt
      721 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
      781 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
      841 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      901 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      961 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     1021 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     1081 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     1141 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     1201 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     1261 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     1321 gggaggtcta tataagcaga gctctctggc taactagaga acccactgct tactggctta
     1381 tcgaaattaa tacgactcac tatagggaga cccaagcttg gtaccgagct cggatcccgc
     1441 tagcaccatg atggatcaag ctagatcagc attctctaac ttgtttggtg gagaaccatt
     1501 gtcatgtacc cgggccagcc tggctcggca agtagatggc gataacagtc atgtggagat
     1561 gaaacttgct gtagatgaag aagaaaatgc tgacaataac acaaaggcca atgtcacaaa
     1621 accaaaaagg tgtagtggaa gtatctgcta tgggactatt gctgtgatcg tctttttctt
     1681 gattggattt atgattggct acttgggcta tggaagcgga ggatccggga tggtgaccac
     1741 actgtccgga ctgtctggag agcagggacc atccggcgac atgaccacag aggaggattc
     1801 tgccacacac atcaagttca gctagaggga cgaggacgga agagagctgg caggagcaac
     1861 catggagctg agggatagct ccggcaagac catcagcaca tggatctccg acggccacgt
     1921 gaaggatttc tacctgtatc ccggcaagta cacctttgtg gagacagcag caccagacgg
     1981 atatgaggtg gcaaccccta tcgagtttac agtgaacgag gacggacagg tgaccgtgga
     2041 tggagaggca acagagggcg atgcacacac cggatctagc ggatctatgc gtaaaggcga
     2101 agagctgttc actggtgtcg tccctattct ggtggaactg gatggtgatg tcaacggtca
     2161 taagttttcc gtgcgtggcg agggtgaagg tgacgcaact aatggtaaac tgacgctgaa
     2221 gttcatctgt actactggta aactgccggt accttggccg actctggtaa cgacgctgac
     2281 ttatggtgtt cagtgctttg ctcgttatcc ggaccatatg aagcagcatg acttcttcaa
     2341 gtccgccatg ccggaaggct atgtgcagga acgcacgatt tcctttaagg atgacggcac
     2401 gtacaaaacg cgtgcggaag tgaaatttga aggcgatacc ctggtaaacc gcattgagct
     2461 gaaaggcatt gactttaaag aagacggcaa tatcctgggc cataagctgg aatacaattt
     2521 taacagccac aatgtttaca tcaccgccga taaacaaaaa aatggcatta aagcgaattt
     2581 taaaattcgc cacaacgtgg aggatggcag cgtgcagctg gctgatcact accagcaaaa
     2641 cactccaatc ggtgatggtc ctgttctgct gccagacaat cactatctga gcacgcaaag
     2701 cgttctgtct aaagatccga acgagaaacg cgatcatatg gttctgctgg agttcgtaac
     2761 cgcagcgggc atcacgcatg gtatggatga actatacaaa gaacaaaaac tcatctcaga
     2821 agaggatctg ggttccggtg agcccgaggc ctgatgagaa ttctgcagat atccatcaca
     2881 ctggcggccg ctcgagcatg catctagagg gccctattct atagtgtcac ctaaatgcta
     2941 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3001 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3061 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3121 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     3181 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     3241 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3301 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3361 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3421 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     3481 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3541 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3601 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3661 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3721 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     3781 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3841 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3901 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     3961 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     4021 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     4081 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4141 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4201 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     4261 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4321 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4381 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4441 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4501 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4561 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     4621 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4681 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4741 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4801 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4861 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     4921 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4981 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     5041 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     5101 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     5161 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     5221 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5281 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5341 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5401 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5461 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5521 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5581 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5641 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5701 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5761 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5821 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5881 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5941 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6001 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6061 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6121 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6181 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6241 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6301 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6361 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6421 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6481 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6541 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6601 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6661 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6721 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6781 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6841 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6901 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6961 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7021 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7081 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7141 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7201 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7261 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7321 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
