LOCUS       sequence-344145-        9103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  amber suppression mediated CRFR1 A95TAG expression in mammalian 
            cells; transient or piggy bac mediated integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9103)
  AUTHORS   Meineke B, Heimgartner J, Craig AJ, Landreh M, Moodie LWK, Elsasser 
            SJ
  TITLE     A Genetically Encoded Picolyl Azide for Improved Live Cell Copper 
            Click Labeling.
  JOURNAL   Front Chem. 2021 Nov 11;9:768535. doi: 10.3389/fchem.2021.768535. 
            eCollection 2021.
  PUBMED    34858945
REFERENCE   2  (bases 1 to 9103)
  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: "Front 
            Chem."; date: "2021-11-11"; volume: "9:768535. doi"; pages: " 
            10.3389/fchem.2021.768535. eCollection 2021"
COMMENT     Sequence Label: pAS_4xMmaPylT_EF1_CRFR1-A95TAG-HA
FEATURES             Location/Qualifiers
     source          1..9103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(91..546)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(228..249)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     440..459
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     673..695
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     687..704
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     688..704
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   834..896
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2687..2898
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2911..3179
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      3211..3220
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4462..4488
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(4508..5082,5083..5085,5086..5094)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4675..4692)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4902..4921
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5107..5505
                     /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(5571..5591)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5633..5754
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5670..5689)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5724..5743
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6517..6551
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7042..7058)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7042..7058)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7055..7077)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7066..7082
                     /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(7090..7096,7097..7114,7115..7120))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7135..7156
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7273..7290)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7444..8032)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7524..7543)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8203..8994,8995..9063))
                     /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     8826..8845
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(9064..9103,1..65))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
       61 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
      121 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
      181 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
      241 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
      301 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
      361 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
      421 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
      481 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
      541 gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
      601 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
      661 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcctcgttca
      721 ttcacgtttt tgaacccgtg gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg
      781 tgatggagca gatgaagatg ctcgacacgc tgcagaacac gcagctagat taaccctaga
      841 aagataatca tattgtgacg tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt
      901 ttatcggtct gtatatcgag gtttatttat taatttgaat agatattaag ttttattata
      961 tttacactta catactaata ataaattcaa caaacaattt atttatgttt atttatttat
     1021 taaaaaaaac aaaaactcaa aatttcttct ataaagtaac aaaactttta tgagggacag
     1081 ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg
     1141 agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg
     1201 cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg
     1261 ctgctctttg agcctgcaga cacctggggg gatacgggga aaaggcctcc acggccaact
     1321 agtgaaaaac cgcacttgtc cggaaacccc gggaatctaa cccggctgaa cggatttaga
     1381 gtccgttcga tctacatgat caggtttccg aggtacccag gcggcgcaca agctatataa
     1441 acctgaagga aatctcaact ttacacttag gtcaagttac ttatcgtact agagcttcag
     1501 caggaaattt aactaaaatc taatttaacc agcatagcaa atatcattta ttcccaaaat
     1561 gctaaagttt gagataaacg gacttgattt ccggctgttt tgacactatc cagaatgcct
     1621 tgcagatggg tggggcatgc taaatactgc aggaaaaacc gcacttgtcc ggaaaccccg
     1681 ggaatctaac ccggctgaac ggatttagag tccgttcgat ctacatgatc aggtttccga
     1741 ggtacccagg cggcgcacaa gctatataaa cctgaaggaa atctcaactt tacacttagg
     1801 tcaagttact tatcgtacta gagcttcagc aggaaattta actaaaatct aatttaacca
     1861 gcatagcaaa tatcatttat tcccaaaatg ctaaagtttg agataaacgg acttgatttc
     1921 cggctgtttt gacactatcc agaatgcctt gcagatgggt ggggcatgct aaatactgca
     1981 ggaaaaaccg cacttgtccg gaaaccccgg gaatctaacc cggctgaacg gatttagagt
     2041 ccgttcgatc tacatgatca ggtttccgag gtacccaggc ggcgcacaag ctatataaac
     2101 ctgaaggaaa tctcaacttt acacttaggt caagttactt atcgtactag agcttcagca
     2161 ggaaatttaa ctaaaatcta atttaaccag catagcaaat atcatttatt cccaaaatgc
     2221 taaagtttga gataaacgga cttgatttcc ggctgttttg acactatcca gaatgccttg
     2281 cagatgggtg gggcatgcta aatactgcag gaaaaaccgc acttgtccgg aaaccccggg
     2341 aatctaaccc ggctgaacgg atttagagtc cgttcgatct acatgatcag gtttccgagg
     2401 tacccaggcg gcgcacaagc tatataaacc tgaaggaaat ctcaacttta cacttaggtc
     2461 aagttactta tcgtactaga gcttcagcag gaaatttaac taaaatctaa tttaaccagc
     2521 atagcaaata tcatttattc ccaaaatgct aaagtttgag ataaacggac ttgatttccg
     2581 gctgttttga cactatccag aatgccttgc agatgggtgg ggcatgctaa atactgcaga
     2641 actagtgctc tagaaaggat ctgcgatcgc tccggtgccc gtcagtgggc agagcgcaca
     2701 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg tgcctagaga
     2761 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     2821 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     2881 tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc acgcgcccgc
     2941 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt
     3001 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc
     3061 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg
     3121 accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg ttacagatcc
     3181 aagctgtgac cggcgcctac tctagagcta gccaccatgg gacggcgccc gcagctccgg
     3241 ctcgtgaagg cccttctcct tctggggctg aaccctgtgt ccacctccct tcaggatcag
     3301 cgctgtgaga acctgtccct gaccagcaat gtttctggcc tgcagtgcaa tgcatccgtg
     3361 gacctcattg gcacctgctg gccccggagc cctgcgggcc agttggtggt ccgaccctgc
     3421 cctgcctttt tctacggtgt ccgctacaac acgacaaaca atggctaccg ggagtgcctg
     3481 gccaacggca gctgggcata gcgtgtgaat tattctgagt gccaggagat tctcaacgaa
     3541 gagaagaaga gcaaagtaca ctaccatgtt gcagtcatca tcaactacct gggtcactgc
     3601 atctccctgg tagccctcct ggtggccttt gtcctcttct tgcggctcag gagcatccgg
     3661 tgcctgagaa acatcatcca ctggaacctc atctcggctt tcatcctacg caacgccacg
     3721 tggtttgtgg tccagctcac cgtgagcccc gaggtgcacc agagcaatgt ggcctggtgt
     3781 aggttggtga cagccgccta caattacttc catgtaacca acttcttctg gatgttcggt
     3841 gagggctgct acctgcacac agccattgtg ctcacgtact ccaccgaccg tctgcgcaag
     3901 tggatgttcg tctgcattgg ctggggtgta cctttcccca tcattgtggc ttgggccatt
     3961 gggaagctgc actacgacaa tgaaaagtgc tggtttggca aacgtcctgg ggtatacact
     4021 gactacatct accagggccc catgatcctg gtcctgctga tcaactttat ctttctcttc
     4081 aacattgtcc gcatcctcat gaccaaactc cgggcatcca ctacatctga gaccattcag
     4141 tacaggaagg ctgtgaaggc cactctggtg ctcctgcccc ttctgggcat cacctacatg
     4201 ttgttcttcg tcaaccctgg agaggacgag gtctccaggg tcgtcttcat ctacttcaac
     4261 tcttttctgg agtcctttca gggcttcttt gtgtctgtgt tctactgttt tctgaacagt
     4321 gaggtccgct ccgctatccg gaagaggtgg cgtcggtggc aggacaagca ctccatcaga
     4381 gcccgagtgg cccgagctat gtccatcccc acctccccga ccagagtcag ctttcacagc
     4441 atcaagcagt ccacagcagt gtacccatac gatgttccag attacgcatg aaggatccgc
     4501 ggccgcgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag
     4561 gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga
     4621 gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg
     4681 ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt
     4741 gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca
     4801 ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc
     4861 agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat
     4921 tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc
     4981 ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga
     5041 accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggcg
     5101 tccggaatgg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag agcaacggct
     5161 acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc tctctctagc
     5221 gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa
     5281 ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg tatcgtcgcg
     5341 atcggaaatg agaacagggg catcttgagc ccctgcggac ggtgccgaca ggtgcttctc
     5401 gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc gacggcagtt
     5461 gggattcgtg aattgctgcc ctctggttat gtgtgggagg gctgaccatg ggtcgacaat
     5521 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     5581 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgcgttaac taaacttgtt
     5641 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5701 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     5761 ctggaattga ctcaaatgat gtcaattagt ctatcagaag ctcatctggt ctcccttccg
     5821 ggggacaaga catccctgtt taatatttaa acagcagtgt tcccaaactg ggttcttata
     5881 tcccttgctc tggtcaacca ggttgcaggg tttcctgtcc tcacaggaac gaagtcccta
     5941 aagaaacagt ggcagccagg tttagccccg gaattgactg gattcctttt ttagggccca
     6001 ttggtatggc tttttccccg tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa
     6061 gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct
     6121 gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc ggctcgctgc
     6181 tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg ggggctgtcc
     6241 ctgatatcta taacaagaaa atatatatat aataagttat cacgtaagta gaacatgaaa
     6301 taacaatata attatcgtat gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt
     6361 cattttgact cacgcggtcg ttatagttca aaatcagtga cacttaccgc attgacaagc
     6421 acgcctcacg ggagctccaa gcggcgactg agatgtccta aatgcacagc gacggattcg
     6481 cgctatttag aaagagagag caatatttca agaatgcatg cgtcaatttt acgcagacta
     6541 tctttctagg gttaatctag ctgcatcagg atcatatcgt cgggtctttt ttccggctca
     6601 gtcatcgccc aagctggcgc tatctgggca tcggggagga agaagcccgt gccttttccc
     6661 gcgaggttga agcggcatgg aaagagtttg ccgaggatga ctgctgctgc attgacgttg
     6721 agcgaaaacg cacgtttacc atgatgattc gggaaggtgt ggccatgcac gcctttaacg
     6781 gtgaactgtt cgttcaggcc acctgggata ccagttcgtc gcggcttttc cggacacagt
     6841 tccggatggt cagcccgaag cgcatcagca acccgaacaa taccggcgac agccggaact
     6901 gccgtgccgg tgtgcagatt aatgacagcg gtgcggcgct gggatattac gtcagcgagg
     6961 acgggtatcc tggctggatg ccgcagaaat ggacatggat accccgtgag ttacccggcg
     7021 ggcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     7081 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     7141 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     7201 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     7261 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     7321 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     7381 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     7441 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     7501 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     7561 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     7621 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     7681 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     7741 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     7801 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     7861 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     7921 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     7981 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     8041 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     8101 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     8161 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     8221 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     8281 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     8341 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     8401 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     8461 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     8521 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     8581 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     8641 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     8701 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     8761 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     8821 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     8881 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     8941 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     9001 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     9061 catactcttc ctttttcaat attattgaag catttatcag ggt
//
