LOCUS       sequence-309622-        7226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid with amber suppression reporter sfGFP 150 TAG stop, for 
            transient transfection or stable, piggybac-mediated, integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7226)
  AUTHORS   Meineke B, Heimgartner J, Lafranchi L, Elsasser SJ
  TITLE     Methanomethylophilus alvus Mx1201 provides basis for mutual 
            orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in 
            mammalian cells.
  JOURNAL   ACS Chem Biol. 2018 Sep 27. doi: 10.1021/acschembio.8b00571.
  PUBMED    30260624
REFERENCE   2  (bases 1 to 7226)
  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: "ACS Chem 
            Biol. 2018 Sep 27. doi: 10.1021/acschembio.8b00571."
COMMENT     Sequence Label: pAS_sfGPF 150TAG
FEATURES             Location/Qualifiers
     source          1..7226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     127..149
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     141..158
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     142..158
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   288..350
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        818..1029
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1042..1310
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      1344..1353
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(2085..2659,2660..2662,2663..2671)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2252..2269)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2479..2498
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2684..3082
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(3148..3168)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3210..3331
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3247..3266)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3301..3320
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4094..4128
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4619..4635)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4619..4635)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4632..4654)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4643..4659
                     /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(4667..4673,4674..4691,4692..4697))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4712..4733
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4850..4867)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5021..5609)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5101..5120)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5780..6571,6572..6640))
                     /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     6403..6422
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6641..6745)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6771..7226)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6908..6929)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7120..7139
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
       61 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      121 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcctcg ttcattcacg
      181 tttttgaacc cgtggaggac gggcagactc gcggtgcaaa tgtgttttac agcgtgatgg
      241 agcagatgaa gatgctcgac acgctgcaga acacgcagct agattaaccc tagaaagata
      301 atcatattgt gacgtacgtt aaagataatc atgtgtaaaa ttgacgcatg tgttttatcg
      361 gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat tatatttaca
      421 cttacatact aataataaat tcaacaaaca atttatttat gtttatttat ttattaaaaa
      481 aaacaaaaac tcaaaatttc ttctataaag taacaaaact tttatgaggg acagcccccc
      541 cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc agcgagccgc
      601 ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga
      661 cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt ctcgctgctc
      721 tttgagcctg cagacacctg gggggatacg gggaaaaggc ctccacggcc aactagtgct
      781 ctagaaagga tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
      841 gtccccgaga agttgggggg aggggtcggc aattgaacgg gtgcctagag aaggtggcgc
      901 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
      961 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     1021 agaacacagc tgaagcttcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc
     1081 tgaggccgcc atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc
     1141 tgaactgcgt ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg
     1201 gcgctccctt ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt
     1261 gctcaactct acgtctttgt ttcgttttct gttctgcgcc gttacagatc caagctgtga
     1321 ccggcgccta ctctagagct agcgccacca tggtgtccaa gggcgaggaa ctgttcaccg
     1381 gcgtggtgcc catcctggtg gaactggatg gcgacgtgaa cggccacaag ttctctgtgc
     1441 ggggagaggg cgaaggcgac gccacaaatg gcaagctgac cctggagttc atctgcacca
     1501 ccggcaagct gcccgtgcct tggcctaccc tcgtgaccac actgacctac ggcgtgcagt
     1561 gcttcagcag ataccccgac catatgaagc ggcacgactt cttcaagagc gccatgcccg
     1621 agggctacgt gcaggaacgg accatcagct tcaaggacga cggcacctac aagaccagag
     1681 ccgaagtgaa gttcgagggc gacaccctcg tgaaccggat cgagctgaag ggcatcgatt
     1741 tcaaagagga cggcaacatc ctgggccaca agctggagta caacttcaac agccactagg
     1801 tgtacatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag atccggcaca
     1861 acgtggaaga tggcagcgtg cagctggccg accactacca gcagaacacc cccatcggag
     1921 atggccccgt gctgctgccc gacaaccact acctgagcac ccagagcgtg ctgagcaagg
     1981 accccaacga gaagcgggac cacatggtgc tgctggaatt cgtgaccgcc gctggcatca
     2041 cccacggcat ggacgagctg tacaagggca gctgagcggc cgcgcccctc tccctccccc
     2101 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
     2161 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
     2221 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg
     2281 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     2341 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     2401 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     2461 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     2521 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt
     2581 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
     2641 tgaaaaacac gatgataata tggccacaac catggcgtcc ggaatggcca agcctttgtc
     2701 tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc
     2761 tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt
     2821 caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc
     2881 tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat
     2941 cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc
     3001 catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc
     3061 tggttatgtg tgggagggct gaccatgggt cgacaatcaa cctctggatt acaaaatttg
     3121 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     3181 tttaatgcct ttgtatcatg cgttaactaa acttgtttat tgcagcttat aatggttaca
     3241 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3301 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gaattgactc aaatgatgtc
     3361 aattagtcta tcagaagctc atctggtctc ccttccgggg gacaagacat ccctgtttaa
     3421 tatttaaaca gcagtgttcc caaactgggt tcttatatcc cttgctctgg tcaaccaggt
     3481 tgcagggttt cctgtcctca caggaacgaa gtccctaaag aaacagtggc agccaggttt
     3541 agccccggaa ttgactggat tcctttttta gggcccattg gtatggcttt ttccccgtat
     3601 ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg
     3661 tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga
     3721 gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg
     3781 taattacatc cctgggggct ttgggggggg gctgtccctg atatctataa caagaaaata
     3841 tatatataat aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag
     3901 ttaaatctta aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta
     3961 tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg
     4021 gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa
     4081 tatttcaaga atgcatgcgt caattttacg cagactatct ttctagggtt aatctagctg
     4141 catcaggatc atatcgtcgg gtcttttttc cggctcagtc atcgcccaag ctggcgctat
     4201 ctgggcatcg gggaggaaga agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa
     4261 gagtttgccg aggatgactg ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg
     4321 atgattcggg aaggtgtggc catgcacgcc tttaacggtg aactgttcgt tcaggccacc
     4381 tgggatacca gttcgtcgcg gcttttccgg acacagttcc ggatggtcag cccgaagcgc
     4441 atcagcaacc cgaacaatac cggcgacagc cggaactgcc gtgccggtgt gcagattaat
     4501 gacagcggtg cggcgctggg atattacgtc agcgaggacg ggtatcctgg ctggatgccg
     4561 cagaaatgga catggatacc ccgtgagtta cccggcgggc gcgcttggcg taatcatggt
     4621 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4681 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4741 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4801 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4861 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4921 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4981 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5041 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5101 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5161 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5221 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5281 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5341 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5401 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5461 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5521 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5581 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5641 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5701 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5761 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5821 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5881 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5941 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6001 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6061 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6121 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6181 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6241 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6301 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6361 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6421 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6481 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6541 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6601 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6661 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6721 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     6781 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     6841 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     6901 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     6961 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     7021 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     7081 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     7141 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     7201 gcttaatgcg ccgctacagg gcgcgt
//
