LOCUS       AiRT        7765 bp DNA     circular SYN 28-DEC-2023
DEFINITION  AiRT8  (7765 bp).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7765)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 28, 2023 from SnapGene Viewer 7.0.3
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        913..943
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    1049..1170
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(1183..1216)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    1448..1503
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     CDS             complement(1645..2364)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNIYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFRTAAGITHGMDELYK"
     regulatory      2361..2370
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     enhancer        2529..2832
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2833..3036
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     ncRNA           3163..3189
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           3215..3256
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     regulatory      3364..3373
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3373..3402
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3403..4101
                     /codon_start=1
                     /product="far-red-shifted derivative of red fluorescent 
                     protein from Entacmaea quadricolor, also known as Katushka 
                     (Shcherbo et al., 2007)"
                     /label=TurboFP635
                     /note="mammalian codon-optimized"
                     /translation="GEDSVLITENMHMKLYMEGTVNDHHFKCTSEGEGKPYEGTQTMKI
                     KVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERITTYEDGGVL
                     TATQDTSLQNGCLIYNVKINGVNFPSNGPVMQKKTLGWEASTEMLYPADSGLRGHSQMA
                     LKLVGGGYLHCSLKTTYRSKKPAKNLKMPGFYFVDRRLERIKEADKETYVEQHEMAVAR
                     YCDLPSKLGHS"
     CDS             4156..4221
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4222..4275
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4276..4317
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4318..5028
                     /codon_start=1
                     /product="monomeric green fluorescent protein, also known 
                     as mTagGFP"
                     /label=TagGFP2
                     /note="mammalian codon-optimized"
                     /translation="SGGEELFAGIVPVLIELDGDVHGHKFSVRGEGEGDADYGKLEIKF
                     ICTTGKLPVPWPTLVTTLCYGIQCFARYPEHMKMNDFFKSAMPEGYIQERTIQFQDDGK
                     YKTRGEVKFEGDTLVNRIELKGKDFKEDGNILGHKLEYSFNSHNVYIRPDKANNGLEAN
                     FKTRHNIEGGGVQLADHYQTNVPLGDGPVLIPINHYLSTQTKISKDRNEARDHMVLLES
                     FSACCHTHGMDELYR"
     CDS             5029..5055
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5190..5311
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    5407..5518
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(5660..6259)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     protein_bind    complement(6264..6297)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     polyA_signal    6509..6557
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        6630..6734
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6735..7595
                     /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"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tgggtaccta agatacattg atgagtttgg acaaaccaca
     1081 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     1141 gtaaccatta taagctgcaa taaacaagtt gacggtatcg taataacttc gtatagcata
     1201 cattatacga agttatatgg atccatatgc tagctcgacg ctcaggtcga gggatcttca
     1261 taagagaaga gggacagcta tgactgggag taaggaatgg acagcagggg gctgtttcat
     1321 atactgatga cctctttata gccacctttg ttcatggcag ccagcatatg gcatatgttg
     1381 ccaaactcta aaccaaatac tcattctgat gttttaaatg atttgccctc ccatatgtcc
     1441 ttccgagtga gagacacaaa aaattccaac acactattgc aatgaaaata aatttccttt
     1501 attagccaga agtcagatgc tcaaggggct tcatgatgtc cccataattt ttggcagagg
     1561 gaaaaagatc tcagtggtat ttgtgagcca gggcattggc cacaccagcc accaccttct
     1621 gataggcagc ctgcacctga gtgtttactt gtacagctcg tccatgccgt gagtgatccc
     1681 ggcggcggtg cggaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct
     1741 cagcttggac tgggtgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat
     1801 gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg
     1861 gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg gccatgatat agatgttgtg
     1921 gctgttgaag ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat
     1981 gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt
     2041 cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat
     2101 ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcggctga agcactgcac
     2161 gccgtggctc agggtggtca cgagggtggg ccagggcacg ggcagcttgc cggtggtgca
     2221 gatgaacttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct
     2281 gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa
     2341 cagctcctcg cccttgctca ccatggtggc gaccacgatc ctcatcctgt ctctgcgatc
     2401 tgacggttca ctaaacgagc tctgcttata taggcctccc accgtacacg ccacctcgac
     2461 atactcagta gttattaata gtaatcaatt acggggtcat tagttcatag cgaggctgca
     2521 gtctaccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     2581 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     2641 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     2701 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     2761 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     2821 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     2881 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     2941 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     3001 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagataccg
     3061 gcaggtaagt atcaaggtta caagacaggt ttaaggagac caatagaaac tgggcttgtc
     3121 gagacagaga agactcttgc gtttcggtcg acttctagag ctctggaggc ttgctgaagg
     3181 ctgtatgcta gagacgtaca gatgcgtctc acaggacaca aggcctgtta ctagcactca
     3241 catggaacaa atggccgaat tcggcgctag ctgctgatat cgcatacgcg tggaccagat
     3301 aggcacctat tggtcttact gacatccact ttgcctttct ctccacaggt gccggtcgcc
     3361 accgccacca tggaacaaaa gctgatttct gaagaagact tgggtgagga tagcgtgctg
     3421 atcaccgaga acatgcacat gaaactgtac atggagggca ccgtgaacga ccaccacttc
     3481 aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gaagatcaag
     3541 gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctaccag cttcatgtac
     3601 ggcagcaaaa cctttatcaa ccacacccag ggcatccccg acttctttaa gcagtccttc
     3661 cctgagggct tcacatggga gaggatcacc acatacgaag acgggggcgt gctgaccgct
     3721 acccaggaca ccagcctcca gaacggctgc ctcatctaca acgtcaagat caacggggtg
     3781 aacttcccat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccagcacc
     3841 gagatgctgt accccgctga cagcggcctg agaggccata gccagatggc cctgaagctc
     3901 gtgggcgggg gctacctgca ctgctccctc aagaccacat acagatccaa gaaacccgct
     3961 aagaacctca agatgcccgg cttctacttc gtggacagga gactggaaag aatcaaggag
     4021 gccgacaaag agacctacgt cgagcagcac gagatggctg tggctcgcta ctgcgacctg
     4081 cctagcaaac tggggcacag cctcgagggt ggttctggtg gttctgccta aggtggttct
     4141 ggtggttctg aattcgacta caaggaccac gacggtgact acaaggacca cgacatcgac
     4201 tacaaggacg acgacgacaa ggagggcaga ggaagtctgc taacatgcgg tgacgtcgag
     4261 gagaatcctg gcccaggtaa gcctatccct aaccctctcc tcggtctcga ttctacgagc
     4321 gggggcgagg agctgttcgc cggcatcgtg cccgtgctga tcgagctgga cggcgacgtg
     4381 cacggccaca agttcagcgt gcgcggcgag ggcgagggcg acgccgacta cggcaagctg
     4441 gagatcaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctggtgacc
     4501 accctctgct acggcatcca gtgcttcgcc cgctaccccg agcacatgaa gatgaacgac
     4561 ttcttcaaga gcgccatgcc cgagggctac atccaggagc gcaccatcca gttccaggac
     4621 gacggcaagt acaagacccg cggcgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     4681 atcgagctga agggcaagga cttcaaggag gacggcaaca tcctgggcca caagctggag
     4741 tacagcttca acagccacaa cgtgtacatc cgccccgaca aggccaacaa cggcctggag
     4801 gctaacttca agacccgcca caacatcgag ggcggcggcg tgcagctggc cgaccactac
     4861 cagaccaacg tgcccctggg cgacggcccc gtgctgatcc ccatcaacca ctacctgagc
     4921 actcagacca agatcagcaa ggaccgcaac gaggcccgcg accacatggt gctcctggag
     4981 tccttcagcg cctgctgcca cacccacggc atggacgagc tgtacaggta cccatacgat
     5041 gttccagatt acgcttaaat agatccaccg gatctagata actgatcata atcagccata
     5101 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     5161 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     5221 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5281 gtggtttgtc caaactcatc aatgtatctt aacgcgaaac tagtgctatt gtcttcccaa
     5341 tcctccccct tgctgtcctg ccccacccca ccccccagaa tagaatgaca cctactcaga
     5401 caatgcgatg caatttcctc attttattag gaaaggacag tgggagtggc accttccagg
     5461 gtcaaggaag gcacggggga ggggcaaaca acagatggct ggcaactaga aggcacagtc
     5521 gaggctgatc agcgagctct agagtcggtg ggcctcgggg gcgggtgcgg ggtcggcggg
     5581 gccgccccgg gtggcttcgg tcggagccat ggggtcgtgc gctcctttcg gtcgggcgct
     5641 gcgggtcgtg gggcgggcgt caggcaccgg gcttgcgggt catgcaccag gtgcgcggtc
     5701 cttcgggcac ctcgacgtcg gcggtgacgg tgaagccgag ccgctcgtag aaggggaggt
     5761 tgcggggcgc ggaggtctcc aggaaggcgg gcaccccggc gcgctcggcc gcctccactc
     5821 cggggagcac gacggcgctg cccagaccct tgccctggtg gtcgggcgac acgccgacgg
     5881 tggccaggaa ccacgcgggc tccttgggcc ggtgcggcgc caggaggcct tccatctgtt
     5941 gctgcgcggc cagccgggaa ccgctcaact cggccatgcg cgggccgatc tcggcgaaca
     6001 ccgcccccgc ttcgacgctc tccggcgtgg tccagaccgc caccgcggcg ccgtcgtccg
     6061 cgacccacac cttgccgatg tcgagcccga cgcgcgtgag gaagagttct tgcagctcgg
     6121 tgacccgctc gatgtggcgg tccgggtcga cggtgtggcg cgtggcgggg tagtcggcga
     6181 acgcggcggc gagggtgcgt acggcccggg ggacgtcgtc gcgggtggcg aggcgcaccg
     6241 tgggcttgta ctcggtcatg gtgataactt cgtataggat actttatacg aagttattta
     6301 ttctcgaatg ggggaccccg tccctaaccc acggggccag tggctatggc agggcctgcc
     6361 gccccgacgt tggctgcgag ccctgggcct tcacccgaac ttggggggtg gggtggggaa
     6421 aaggaagaaa cgcgggcgta ttggccccaa tggggtctcg gtggggtatc gacagagtgc
     6481 cagccctggg accgaacccc gcgtttatga acaaacgacc caacacccgt gcgttttatt
     6541 ctgtcttttt attgccgtca tagcgcgggt tccttccggt attgtctcct tccgtgttta
     6601 ttaagcttgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     6661 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     6721 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     6781 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     6841 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     6901 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     6961 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     7021 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     7081 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     7141 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     7201 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     7261 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     7321 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     7381 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     7441 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     7501 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     7561 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     7621 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     7681 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     7741 ccgtagaaaa gatcaaagga tcttc
//
