LOCUS       sequence-380314-        7226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pAS control plasmid for sfGFP expression .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7226)
  AUTHORS   Meineke B, Elsasser SJ
  TITLE     Generation of Amber Suppression Cell Lines Using CRISPR-Cas9.
  JOURNAL   Methods Mol Biol. 2023;2676:169-180. doi: 
            10.1007/978-1-0716-3251-2_12.
  PUBMED    37277632
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: "Methods Mol
            Biol."; date: "2023"; volume: "2676:169-180. doi"; pages: " 
            10.1007/978-1-0716-3251-2_12"
COMMENT     Sequence Label: pAS_sfGFP150K
FEATURES             Location/Qualifiers
     source          1..7226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(534..1325,1326..1394))
                     /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     1157..1176
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1395..1499)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1525..1980)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1662..1683)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1874..1893
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2107..2129
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2121..2138
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2122..2138
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   2268..2330
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2798..3009
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             3022..3290
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      3324..3333
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(4065..4639,4640..4642,4643..4651)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4232..4249)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4459..4478
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4664..5062
                     /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(5128..5148)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5190..5311
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5227..5246)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5281..5300
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6074..6108
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6599..6615)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6599..6615)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6612..6634)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6623..6639
                     /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(6647..6653,6654..6671,6672..6677))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6692..6713
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6830..6847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(7001..7226,1..363))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7081..7100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
       61 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
      121 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
      181 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
      241 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
      301 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
      361 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
      421 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
      481 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
      541 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
      601 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
      661 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
      721 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
      781 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
      841 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
      901 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
      961 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     1021 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     1081 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     1141 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     1201 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     1261 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     1321 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     1381 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     1441 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     1501 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     1561 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     1621 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     1681 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     1741 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     1801 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     1861 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     1921 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     1981 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     2041 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     2101 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcctcg ttcattcacg
     2161 tttttgaacc cgtggaggac gggcagactc gcggtgcaaa tgtgttttac agcgtgatgg
     2221 agcagatgaa gatgctcgac acgctgcaga acacgcagct agattaaccc tagaaagata
     2281 atcatattgt gacgtacgtt aaagataatc atgtgtaaaa ttgacgcatg tgttttatcg
     2341 gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat tatatttaca
     2401 cttacatact aataataaat tcaacaaaca atttatttat gtttatttat ttattaaaaa
     2461 aaacaaaaac tcaaaatttc ttctataaag taacaaaact tttatgaggg acagcccccc
     2521 cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc agcgagccgc
     2581 ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga
     2641 cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt ctcgctgctc
     2701 tttgagcctg cagacacctg gggggatacg gggaaaaggc ctccacggcc aactagtgct
     2761 ctagaaagga tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
     2821 gtccccgaga agttgggggg aggggtcggc aattgaacgg gtgcctagag aaggtggcgc
     2881 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     2941 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     3001 agaacacagc tgaagcttcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc
     3061 tgaggccgcc atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc
     3121 tgaactgcgt ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg
     3181 gcgctccctt ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt
     3241 gctcaactct acgtctttgt ttcgttttct gttctgcgcc gttacagatc caagctgtga
     3301 ccggcgccta ctctagagct agcgccacca tggtgtccaa gggcgaggaa ctgttcaccg
     3361 gcgtggtgcc catcctggtg gaactggatg gcgacgtgaa cggccacaag ttctctgtgc
     3421 ggggagaggg cgaaggcgac gccacaaatg gcaagctgac cctgaagttc atctgcacca
     3481 ccggcaagct gcccgtgcct tggcctaccc tcgtgaccac actgacctac ggcgtgcagt
     3541 gcttcagcag ataccccgac catatgaagc ggcacgactt cttcaagagc gccatgcccg
     3601 agggctacgt gcaggaacgg accatcagct tcaaggacga cggcacctac aagaccagag
     3661 ccgaagtgaa gttcgagggc gacaccctcg tgaaccggat cgagctgaag ggcatcgatt
     3721 tcaaagagga cggcaacatc ctgggccaca agctggagta caacttcaac agccacaagg
     3781 tgtacatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag atccggcaca
     3841 acgtggaaga tggcagcgtg cagctggccg accactacca gcagaacacc cccatcggag
     3901 atggccccgt gctgctgccc gacaaccact acctgagcac ccagagcgtg ctgagcaagg
     3961 accccaacga gaagcgggac cacatggtgc tgctggaatt cgtgaccgcc gctggcatca
     4021 cccacggcat ggacgagctg tacaagggca gctgagcggc cgcgcccctc tccctccccc
     4081 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
     4141 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
     4201 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg
     4261 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     4321 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     4381 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     4441 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     4501 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt
     4561 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
     4621 tgaaaaacac gatgataata tggccacaac catggcgtcc ggaatggcca agcctttgtc
     4681 tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc
     4741 tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt
     4801 caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc
     4861 tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat
     4921 cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc
     4981 catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc
     5041 tggttatgtg tgggagggct gaccatgggt cgacaatcaa cctctggatt acaaaatttg
     5101 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     5161 tttaatgcct ttgtatcatg cgttaactaa acttgtttat tgcagcttat aatggttaca
     5221 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5281 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gaattgactc aaatgatgtc
     5341 aattagtcta tcagaagctc atctggtctc ccttccgggg gacaagacat ccctgtttaa
     5401 tatttaaaca gcagtgttcc caaactgggt tcttatatcc cttgctctgg tcaaccaggt
     5461 tgcagggttt cctgtcctca caggaacgaa gtccctaaag aaacagtggc agccaggttt
     5521 agccccggaa ttgactggat tcctttttta gggcccattg gtatggcttt ttccccgtat
     5581 ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg
     5641 tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga
     5701 gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg
     5761 taattacatc cctgggggct ttgggggggg gctgtccctg atatctataa caagaaaata
     5821 tatatataat aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag
     5881 ttaaatctta aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta
     5941 tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg
     6001 gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa
     6061 tatttcaaga atgcatgcgt caattttacg cagactatct ttctagggtt aatctagctg
     6121 catcaggatc atatcgtcgg gtcttttttc cggctcagtc atcgcccaag ctggcgctat
     6181 ctgggcatcg gggaggaaga agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa
     6241 gagtttgccg aggatgactg ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg
     6301 atgattcggg aaggtgtggc catgcacgcc tttaacggtg aactgttcgt tcaggccacc
     6361 tgggatacca gttcgtcgcg gcttttccgg acacagttcc ggatggtcag cccgaagcgc
     6421 atcagcaacc cgaacaatac cggcgacagc cggaactgcc gtgccggtgt gcagattaat
     6481 gacagcggtg cggcgctggg atattacgtc agcgaggacg ggtatcctgg ctggatgccg
     6541 cagaaatgga catggatacc ccgtgagtta cccggcgggc gcgcttggcg taatcatggt
     6601 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     6661 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     6721 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6781 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     6841 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     6901 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6961 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     7021 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     7081 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     7141 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     7201 cacgctgtag gtatctcagt tcggtg
//
