LOCUS       sequence-409233-        7865 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of heterobifunctional SasG linkers (4G).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7865)
  AUTHORS   Sigmund F, Berezin O, Beliakova S, Magerl B, Drawitsch M, Piovesan 
            A, Goncalves F, Bodea SV, Winkler S, Bousraou Z, Grosshauser M, 
            Samara E, Pujol-Marti J, Schadler S, So C, Irsen S, Walch A, Kofler 
            F, Piraud M, Kornfeld J, Briggman K, Westmeyer GG
  TITLE     Genetically encoded barcodes for correlative volume electron 
            microscopy.
  JOURNAL   Nat Biotechnol. 2023 Apr 17. doi: 10.1038/s41587-023-01713-y.
  PUBMED    37069313
REFERENCE   2  (bases 1 to 7865)
  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"; journalName: "Nat 
            Biotechnol. 2023 Apr 17. doi: 10.1038/s41587-023-01713-y."
COMMENT     Sequence Label: sfGFP-SasG(4G)-mCherry
FEATURES             Location/Qualifiers
     source          1..7865
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(964..966,967..1677)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3873..3890)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3879..4103
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4149..4577
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4236..4255)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4446..4467
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4586..4606)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4591..4920
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4771..4906
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4833..4852
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4968..5015
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5034..5408
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    5538..5659
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5575..5594)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5629..5648
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5708..5724)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5708..5724)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5721..5743)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5732..5748
                     /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(5756..5762,5763..5780,5781..5786))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5801..5822
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5939..5956)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6110..6698)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6190..6209)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6869..7660,7661..7729))
                     /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     7492..7511
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7730..7834)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattcgcc
      961 accatgagca agggcgaaga actgtttacc ggcgtggtgc ccatcctggt ggaactggat
     1021 ggggatgtga acggccacaa gttcagcgtc agaggcgaag gcgaagggga tgccacaaac
     1081 ggcaagctga ccctgaagtt catctgcacc accggaaagc tgcccgtgcc ttggcctaca
     1141 ctggtcacca cactgacata cggcgtgcag tgcttcagca gataccccga ccatatgaag
     1201 cggcacgact tcttcaagag cgccatgcct gagggctacg tgcaagagag aaccatcagc
     1261 ttcaaggacg acggcaccta caagaccaga gccgaagtga agttcgaggg cgacaccctg
     1321 gtcaaccgga tcgagctgaa gggcatcgat ttcaaagagg acggcaacat cctgggccac
     1381 aagcttgagt acaacttcaa cagccacaac gtgtacatca ccgccgacaa gcagaagaac
     1441 ggcatcaagg ccaacttcaa gatccggcac aacgtggaag atggcagcgt gcagctggcc
     1501 gatcactacc agcagaacac acccatcgga gatggccctg tgctgctgcc cgataaccac
     1561 tacctgagca cacagagcgt gctgagcaag gaccccaacg agaagaggga ccacatggtg
     1621 ctgctggagt ttgtgacagc cgccggaatc acccacggca tggacgaact ttacaaaggc
     1681 ggcaccggtg gcagcgcccc taagacaatt accgagctgg aaaagaaggt ggaagagatc
     1741 cccttcaaga aagagcggaa gttcaacccc gatctggccc ctggcaccga gaaagtgaca
     1801 agagaaggcc agaagggcga gaaaaccatc accacaccta cactgaagaa ccctctgacc
     1861 ggcgtgatca tctccaaggg cgagcccaaa gaggaaatca caaaggaccc tatcaacgag
     1921 ctgaccgagt acggccccga gacaattgct cctggccaca gagatgagtt cgaccccaaa
     1981 ctgcccaccg gcgagaaaga agaagtgcct ggcaagcctg gcatcaagaa ccctgagaca
     2041 ggcgacgtcg tcagacctcc tgtggacagc gtgaccaaat acggccctgt gaagggcgac
     2101 agcatcgtgg aaaaagaaga gattccgttt gagaaagaga gaaagtttaa tcccgacctc
     2161 gctccaggga cagaaaaagt cactcgcgag ggccagaaag gggaaaagac aatcacaacc
     2221 ccaacgctga aaaatcccct gaccggggag attatcagca aaggcgagag caaagaagaa
     2281 attacgaaag atcccatcaa tgaactcaca gagtacgggc ccgagacaat tgctcctggc
     2341 cacagagatg agttcgaccc caaactgccc accggcgaga aagaagaagt gcctggcaag
     2401 cctggcatca agaaccctga gacaggcgac gtcgtcagac ctcctgtgga cagcgtgacc
     2461 aaatacggcc ctgtgaaggg cgacagcatc gtggaaaaag aagagattcc gtttgagaaa
     2521 gagagaaagt ttaatcccga cctcgctcca gggacagaaa aagtcactcg cgagggccag
     2581 aaaggggaaa agacaatcac aaccccaacg ctgaaaaatc ccctgaccgg ggagattatc
     2641 agcaaaggcg agagcaaaga agaaattacg aaagatccca tcaatgaact cacagagtac
     2701 gggcccgaga caattgctcc tggccacaga gatgagttcg accccaaact gcccaccggc
     2761 gagaaagaag aagtgcctgg caagcctggc atcaagaacc ctgagacagg cgacgtcgtc
     2821 agacctcctg tggacagcgt gaccaaatac ggccctgtga agggcgacag catcgtggaa
     2881 aaagaagaga ttccgtttga gaaagagaga aagtttaatc ccgacctcgc tccagggaca
     2941 gaaaaagtca ctcgcgaggg ccagaaaggg gaaaagacaa tcacaacccc aacgctgaaa
     3001 aatcccctga ccggggagat tatcagcaaa ggcgagagca aagaagaaat tacgaaagat
     3061 cccatcaatg aactcacaga gtacgggcct gagacgggct ccggagccac aggatctgtg
     3121 tccaaaggcg aagaggacaa catggccatc atcaaagaat ttatgcggtt caaggtgcac
     3181 atggaaggct ccgtgaatgg ccacgagttt gagattgaag gcgaaggcga gggcagaccc
     3241 tatgagggaa cacagaccgc caagctgaaa gtgaccaaag gcggacctct gcctttcgcc
     3301 tgggacattc tgagccctca gtttatgtac ggctccaagg cctacgtgaa gcaccccgcc
     3361 gatattcccg actacctgaa gctgagcttc cccgagggct tcaagtggga gagagtgatg
     3421 aatttcgagg acggcggcgt ggtcaccgtg acacaagata gttctctgca ggacggcgag
     3481 ttcatctaca aagtgaagct gcggggcacc aactttccct ctgacggacc cgtgatgcag
     3541 aaaaagacca tgggctggga agccagcagc gagagaatgt accctgaaga tggcgccctg
     3601 aaaggggaga tcaagcagcg gctgaaactg aaggatggcg gccactacga cgccgaagtc
     3661 aagaccacat acaaggccaa gaaaccagtg cagctgccag gcgcctacaa tgtgaacatc
     3721 aagctggaca tcaccagcca caatgaggac tacacaatcg tggaacagta cgagagagcc
     3781 gaggggagac actctaccgg cggaatggat gagctgtaca agtgataagc ggccgctcga
     3841 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     3901 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3961 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     4021 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     4081 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     4141 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4201 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4261 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4321 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4381 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4441 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     4501 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     4561 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     4621 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     4681 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4741 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4801 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     4861 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4921 aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat
     4981 catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca
     5041 agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct
     5101 ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc
     5161 gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc
     5221 tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt
     5281 ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg
     5341 ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc
     5401 aggactgaca cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5461 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5521 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5581 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5641 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     5701 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5761 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5821 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5881 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5941 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     6001 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     6061 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6121 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6181 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6241 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6301 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6361 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6421 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6481 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6541 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6601 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6661 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6721 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6781 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6841 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6901 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6961 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     7021 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     7081 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7141 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7201 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7261 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7321 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7381 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7441 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7501 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7561 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7621 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7681 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7741 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7801 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7861 acgtc
//
