LOCUS       sequence-482910-        8380 bp    DNA     circular SYN 12-AUG-2025
DEFINITION  Expression of the tri-cistronic construct 
            NES-AmNeonGreen-P2A-mTurquoise2-NLS-T2A-mScarlet-I-PTS1 in 
            Exaiptasia diaphana; In vitro transcription of the same construct 
            via SP6 polymerase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8380)
  AUTHORS   n/a
  TITLE     Aiptasia Genetic Tools Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8380)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 12, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pCR197
FEATURES             Location/Qualifiers
     source          1..8380
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(163..184)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2965..2983
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2965..2982
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             3773..3829
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(3830..3832,3833..3835,3836..4546)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3875..3896)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4136..4155)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4483..4504
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4553..4573
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4583..4636
                     /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             4637..5332
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(4770..4788)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5038..5057
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    5386..5520
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5410..5429)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5464..5483
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(6215..6235)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6215..6233)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6254..6270)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6254..6270)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6267..6289)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6278..6294
                     /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(6302..6308,6309..6326,6327..6332))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6347..6368
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6485..6502)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6656..7244)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6736..6755)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7415..8206,8207..8275))
                     /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     8038..8057
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8276..8380)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
       61 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
      121 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
      181 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
      241 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
      301 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggccaca
      361 atgacggcta ctacatggtg ccattccttc ctctttatag gaatggagac tacctcctgt
      421 ccaacaatgc tcttggatac gagtacgcct acctgttgga cccaggtcat tgcacaacac
      481 cagaaatgcc ctctgatctg caaaagacgt gaatatctgt tcagacaccc atatccactc
      541 tgttccacac aggtcagagg tttgtccagg agttcttgac agaggtgtaa aaagtactca
      601 aaaattttac tcaagtgaaa gtacaagtac ttagggaaaa ttttactcaa ttaaaagtaa
      661 aagtatctgg ctagaatctt acttgagtaa aagtaaaaaa gtactccatt aaaattgtac
      721 ttgagtatta aggaagtaaa agtaaaagca agaaagaaaa ctagagattc ttgtttaagc
      781 ttttaatctc aaaaaacatt aaatgaaatg catacaaggt tttatcctgc tttagaactg
      841 tttgtattta attatcaaac tataagacag acaatctaat gccagtacac gctactcaaa
      901 gttgtaaaac ctcagattta acttcagtag aagctgattc tcaaaattgt tagtgtcaag
      961 cctagctctt ttggggctga aaagcaatcc tgcagtgctg aaaagcctct cacaggcagc
     1021 cgatgcggga agaggtgtat tagtcttgat agagaggctg caaatagcag gaaacgtgag
     1081 cagagactcc ctggtgtctg aaacacaggc cagataggcc ccttacgtac gctagcgata
     1141 tcggatccgt cgactgcatt cactgttctt aatattgtat aataggcaaa aatcgaggtc
     1201 acagatctcg aagaaaattg gtaaaataaa caaaatagag aataattaat ctaatgaatc
     1261 gaaaaactcg ttttcagaca ttcaaataag aaacaaaatg tcacataatg gatataaaca
     1321 agtcaatcac aatgcaaaac ctccatcacc ggaggctccg gtcggattgt gtgagatggg
     1381 aagggtacaa aggctcatgg gtgtctatag caagtttaca cagggatccc aaatatattt
     1441 atactctttt aactgtttgg atttattatt cttgactttc acatgacgtc atcagtcaaa
     1501 aaaaaaatct aaaatcacag acacacaaca ttgtggtact gaaatatctt gaagagcatg
     1561 atagaagttc gctctgaatg tttaaacttt ttttgctgtt tattcattga gttattagcg
     1621 ttcaaaaata tgttacgtaa tactgccacg cgacttgata cacgccaaaa atactcgaaa
     1681 agcctatgca tcttaaaaca attatagttt cagcatcaat ttaaatgtta acaatgatat
     1741 tgttgtaaag ttaaagcaaa atcatcagtt ttgagctcaa ttcgatacgg tccgccattt
     1801 tgaagcccct gacggggaca caatcatggc ggccccatac atttctttgt aaagtaagac
     1861 cgtattcaaa cgcttacaat tttgctagga aatcacagtt ttgattcaaa ctttacataa
     1921 ctgttgggag ttatatactt ttttgatccc tgaacttaaa aaattaattt gaatcttaaa
     1981 acaaagcttt ttgtagtcat ttcttgatca gtccaaaacg cattgagccg tgctaatccc
     2041 atacattctt accaattttc gatcgctcgt atttctgtta aaatttatta tttcggctca
     2101 aaatttcgtc atgatcctta aattaagatg ttctctcgag agcattaata aaagtttgct
     2161 aattctgcct ctaaataggt tttttagact atcacgtgat cagtgaaagt caagaattga
     2221 ttggctatga aattataaga aactatgata tttattagtt ttttggtaag ctgaaattct
     2281 aaagcaatca aaatattttt cttgtctgcc agcgccattt ttttcgataa gtcactttgt
     2341 cgaagaaaga agaaacacct aagatagaat ctaaaatcaa aagcaaaaac tactagagct
     2401 atgctaaagc caagaagttt gcaaaaaaac ttgtgaatat taaaattatt gaaattgaaa
     2461 ttaacttcgg cctttttcag gcctttgaaa acgcaaacaa aagccagtaa atgaaccaga
     2521 aaagacgcca agaaactaac tttgtttgct atggtcatta ttcttaattg aaaacatatg
     2581 aaatcacaat ttagcatact ttttgtctac ttttgttcgg agtctggaca gcgtgctctg
     2641 tcacgctgta agctgtgact cttttgttac atctccattg ttgaactgtc aagtgtttcg
     2701 tagcaaccca agtcttcaat ttgtgattgg tttattcatt cttccgtagc gatatttaag
     2761 acaacgcatt cttaatcacc gcctctttta catcatccaa ggccacgtgt gcaacttgtg
     2821 agtttttctc ttacttttac tgcccttttc tccccttttt aactcaaatt atcgttactt
     2881 aagcgaattg tgacattcgt tgaactgttt tctttaaaat ttgtatttta gatcactgtt
     2941 gaatcgctcg gcaacttttc aaatatttag gtgacactat agaagcgctg gtaccataat
     3001 ggctatcaat ttgcaagaaa ttgaggatgt ccgcaagaaa ctggacgaac tggacatcga
     3061 tttaatggtg agcaagggcg aggaggataa catggcctct ctcccagcga cacatgagtt
     3121 acacatcttt ggctccatca acggtgtgga ctttgacatg gtgggtcagg gcaccggcaa
     3181 tccaaatgat ggttatgagg agttaaacct aaagtccacc aagggtgacc tccagttctc
     3241 cccctggatt ctcgtccctc atatcgggta tggcttccat cagtacctac cctaccctga
     3301 cgggatgagt cctttccagg ccgctatggt agatggctcc ggataccaag tccatcgtac
     3361 aatgcagttt gaagatggtg cctcccttac tgttaactac agatacacct acgagggaag
     3421 ccacatcaaa ggagaggccc aggtgaaggg gactggtttc cctgctgacg gtcctgtgat
     3481 gaccaacagt ctcaccgctg cggactggtg caggagtaag aagacttacc ccaacgacaa
     3541 aaccatcatc agtaccttta agtggagtta caccactgga aatggcaaga gataccgtag
     3601 cactgcgagg accacctaca cctttgctaa gccaatggcg gctaactatc tcaagaacca
     3661 gcctatgtac gtgttccgta agacggaact caagcactcc aagaccgagc tcaacttcaa
     3721 ggagtggcaa aaggccttta ccgatgtgat gggcatggac gagctgtaca aggccacgaa
     3781 cttctctctg ttaaagcaag caggagacgt ggaagaaaac cccggtccta tggtgagcaa
     3841 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     3901 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     3961 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     4021 cctgtcctgg ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt
     4081 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     4141 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     4201 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     4261 caactacttt agcgacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc
     4321 caacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca
     4381 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     4441 ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     4501 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggat ccccaaagaa
     4561 gaagcgtaag gtagaagacc cagagggcag gggaagtctt ctaacatgcg gggacgtgga
     4621 ggaaaatccc ggccccatgg tgagcaaggg cgaggcagtg atcaaggagt tcatgcggtt
     4681 caaggtgcac atggagggct ccatgaacgg ccacgagttc gagatcgagg gcgagggcga
     4741 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     4801 gcccttctcc tgggacatcc tgtcccctca gttcatgtac ggctccaggg ccttcatcaa
     4861 gcaccccgcc gacatccccg actactataa gcagtccttc cccgagggct tcaagtggga
     4921 gcgcgtgatg aacttcgagg acggcggcgc cgtgaccgtg acccaggaca cctccctgga
     4981 ggacggcacc ctgatctaca aggtgaagct ccgcggcacc aacttccctc ctgacggccc
     5041 cgtaatgcag aagaagacaa tgggctggga agcgtccacc gagcggttgt accccgagga
     5101 cggcgtgctg aagggcgaca ttaagatggc cctgcgcctg aaggacggcg gccgctacct
     5161 ggcggacttc aagaccacct acaaggccaa gaagcccgtg cagatgcccg gcgcctacaa
     5221 cgtcgaccgc aagttggaca tcacctccca caacgaggac tacaccgtgg tggaacagta
     5281 cgaacgctcc gagggccgcc actccaccgg cggcatggac gagctgtaca agagcggaag
     5341 taagctgtag ttgcccatgg catatggtat acatcgatga gctgcgatcc agacatgata
     5401 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     5461 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     5521 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
     5581 taaagcaagt aaaacctcta caaatgtggt atggctgatt atgatccgcg gacgtggcca
     5641 tctagaattc agatctgacc tggcgaagat acggccacgg gtgctcttga tcctgtggct
     5701 gattttggac tgtgctgctc gcagctgctg atgaatcaca tacttcctcc attttcttcc
     5761 actgattgac tgttataatt tccctaattt ccaggtcaag gtgctgtgca ttgtggtaat
     5821 agatgtgaca tgacgtcact tccaaaggac caatgaacat gtctgaccaa tttcatataa
     5881 tgtgaaaacg attttcatag gcagaataaa taacatttaa attaaactgg gcatcagcgc
     5941 aattcaattg gtttggtaat agcaagggaa aatagaatga agtgatctcc aaaaaataag
     6001 tactttttga ctgtaaataa aattgtaagg agtaaaaagt actttttttt ctaaaaaaat
     6061 gtaattaagt aaaagtaaaa gtattgattt ttaattgtac tcaagtaaag taaaaatccc
     6121 caaaaataat acttaagtac agtaatcaag taaaattact caagtacttt acacctctgc
     6181 ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct
     6241 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     6301 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     6361 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6421 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     6481 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     6541 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     6601 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6661 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6721 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6781 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6841 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6901 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6961 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     7021 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     7081 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     7141 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     7201 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     7261 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     7321 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     7381 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     7441 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7501 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     7561 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     7621 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7681 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7741 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7801 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7861 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7921 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7981 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     8041 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     8101 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     8161 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     8221 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     8281 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     8341 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
//
