LOCUS       sequence-409221-        7097 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of heterobifunctional SasG linkers (2G).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7097)
  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 7097)
  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(2G)-mCherry
FEATURES             Location/Qualifiers
     source          1..7097
                     /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(3105..3122)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3111..3335
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3381..3809
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3468..3487)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3678..3699
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3818..3838)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3823..4152
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4003..4138
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4065..4084
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4200..4247
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4266..4640
                     /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    4770..4891
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4807..4826)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4861..4880
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4940..4956)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4940..4956)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4953..4975)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4964..4980
                     /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(4988..4994,4995..5012,5013..5018))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5033..5054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5171..5188)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5342..5930)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5422..5441)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6101..6892,6893..6961))
                     /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     6724..6743
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6962..7066)
                     /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 ctgagacggg ctccggagcc
     2341 acaggatctg tgtccaaagg cgaagaggac aacatggcca tcatcaaaga atttatgcgg
     2401 ttcaaggtgc acatggaagg ctccgtgaat ggccacgagt ttgagattga aggcgaaggc
     2461 gagggcagac cctatgaggg aacacagacc gccaagctga aagtgaccaa aggcggacct
     2521 ctgcctttcg cctgggacat tctgagccct cagtttatgt acggctccaa ggcctacgtg
     2581 aagcaccccg ccgatattcc cgactacctg aagctgagct tccccgaggg cttcaagtgg
     2641 gagagagtga tgaatttcga ggacggcggc gtggtcaccg tgacacaaga tagttctctg
     2701 caggacggcg agttcatcta caaagtgaag ctgcggggca ccaactttcc ctctgacgga
     2761 cccgtgatgc agaaaaagac catgggctgg gaagccagca gcgagagaat gtaccctgaa
     2821 gatggcgccc tgaaagggga gatcaagcag cggctgaaac tgaaggatgg cggccactac
     2881 gacgccgaag tcaagaccac atacaaggcc aagaaaccag tgcagctgcc aggcgcctac
     2941 aatgtgaaca tcaagctgga catcaccagc cacaatgagg actacacaat cgtggaacag
     3001 tacgagagag ccgaggggag acactctacc ggcggaatgg atgagctgta caagtgataa
     3061 gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     3121 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3181 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3241 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3301 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     3361 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3421 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3481 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3541 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3601 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3661 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3721 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3781 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     3841 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3901 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3961 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     4021 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4081 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4141 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg
     4201 ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact
     4261 aaaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag
     4321 cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg
     4381 ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc
     4441 cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt
     4501 cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg
     4561 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg
     4621 tggccgagga gcaggactga cacgtgctac gagatttcga ttccaccgcc gccttctatg
     4681 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4741 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4801 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4861 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4921 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4981 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5041 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5101 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5161 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5221 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5281 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5341 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5401 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5461 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5521 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5581 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5641 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5701 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5761 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5821 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5881 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5941 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6001 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6061 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6121 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6181 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6241 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6301 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6361 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6421 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6481 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6541 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6601 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6661 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6721 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6781 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6841 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6901 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6961 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7021 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7081 aagtgccacc tgacgtc
//
