LOCUS       sequence-477088-        3572 bp    DNA     circular SYN 26-JUN-2025
DEFINITION  BsaI Golden Gate assembly plasmid encoding the rtTA-P2A-iRFP720 CDS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3572)
  AUTHORS   Peterman EL, Ploessl DS, Love KS, Sanabria V, Daniels RF, Johnstone 
            CP, Godavarti DR, Kabaria SR, Oakes CG, Pai AA, Galloway KE
  TITLE     High-resolution profiling reveals coupled transcriptional and 
            translational regulation of transgenes.
  JOURNAL   bioRxiv [Preprint]. 2024 Dec 3:2024.11.26.625483. doi: 
            10.1101/2024.11.26.625483.
  PUBMED    39651241
REFERENCE   2  (bases 1 to 3572)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 26, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Dec 3:2024.11.26.625483. doi: 
            10.1101/2024.11.26.625483."
COMMENT     Sequence Label: pKG3659_pPV2-rtTA-P2A-iRFP720
FEATURES             Location/Qualifiers
     source          1..3572
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      83..671
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     572..591
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             702..1445
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             1467..1523
                     /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             1524..2474
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 720 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP720
                     /note="derived from Rhodopseudomonas palustris RpBphP2"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     promoter        2519..2623
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2624..2692,2693..3484)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALITFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGYRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2842..2861)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
       61 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
      121 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
      181 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
      241 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
      301 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
      361 caagacgata gttaccggat aagtcgcagc ggtcgggctg aacggggggt tcgtgcacac
      421 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
      481 aaagcgccac gcttccagaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
      541 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
      601 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      661 gcctatggaa aaacgccagc aacgggtctc aaatggccac catgagccgg ctggacaaga
      721 gcaaagtgat caacagcgcc ctggaactgc tgaacggcgt gggcatcgag ggcctgacca
      781 cccggaagct ggcccagaaa ctgggcgtgg aacagcccac cctgtactgg cacgtgaaga
      841 acaagcgggc cctgctggac gccctgccca tcgagatgct ggaccggcac cacacccaca
      901 gctgccctct ggaaggcgag agctggcagg acttcctgcg gaacaacgcc aagagctaca
      961 gatgcgccct gctgagccac cgggacggcg ccaaagtgca cctgggcacc agacccaccg
     1021 agaagcagta cgagacactg gaaaaccagc tggccttcct gtgccagcag ggcttcagcc
     1081 tggaaaacgc cctgtacgcc ctgagcgccg tgggccactt caccctgggc tgcgtgctgg
     1141 aagaacagga acaccaggtc gccaaagagg aaagagagac acccaccacc gacagcatgc
     1201 cccccctgct gaagcaggcc atcgagctgt tcgacagaca gggcgccgag cccgccttcc
     1261 tgttcggcct ggaactgatc atctgcggcc tcgagaagca gctgaagtgc gagagcggcg
     1321 gacccaccga cgccctggac gacttcgacc tggacatgct gcccgccgat gccctggatg
     1381 attttgatct ggatatgctc cctgctgacg ctctcgatga cttcgatctc gatatgctgc
     1441 caggcgctag cggccgcgga agcggagcta ctaacttcag cctgctgaag caggctggag
     1501 acgtggagga gaaccctgga cctatggcgg aaggatccgt cgccaggcag cctgacctct
     1561 tgacctgcga cgatgagccg atccatatcc ccggtgccat ccaaccgcat ggactgctgc
     1621 tcgccctcgc cgccgacatg acgatcgttg ccggcagcga caaccttccc gaactcaccg
     1681 gactggcgat cggcgccctg atcggccgct ctgcggccga tgtcttcgac tcggagacgc
     1741 acaaccgtct gacgatcgcc ttggccgagc ccggggcggc cgtcggagca ccgatcactg
     1801 tcggcttcac gatgcgaaag gacgcaggct tcatcggctc ctggcatcgc catgatcagc
     1861 tcatcttcct cgagctcgag cctccccagc gggacgtcgc cgagccgcag gcgttcttcc
     1921 gccgcaccaa cagcgccatc cgccgcctgc aggccgccga aaccttggaa agcgcctgcg
     1981 ccgccgcggc gcaagaggtg cggaagatta ccggcttcga tcgggtgatg atctatcgct
     2041 tcgcctccga cttcagcggg tccgtgatcg cagaggatcg gtgcgccgag gtcgagtcaa
     2101 aactaggcct gcactatcct gcctcattca tcccggcgca ggcccgtcgg ctctatacca
     2161 tcaacccggt acggatcatt cccgatatca attatcggcc ggtgccggtc accccagacc
     2221 tcaatccggt caccgggcgg ccgattgatc ttagcttcgc catcctgcgc agcgtctcgc
     2281 ccaaccatct ggagttcatg cgcaacatag gcatgcacgg cacgatgtcg atctcgattt
     2341 tgcgcggcga gcgactgtgg ggattgatcg tttgccatca ccgaacgccg tactacgtcg
     2401 atctcgatgg ccgccaagcc tgcgagctag tcgcccaggt tctggcctgg cagatcggcg
     2461 tgatggaaga gtaacaacag agacctagac gtcaggtggc acttttcggg gaaatgtgcg
     2521 cggaacccct atttgtttat ttttataaat acattcaaat atgtatccgc tcatgagaca
     2581 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2641 ccgtgtcgcc cttattacct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2701 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2761 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2821 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca
     2881 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2941 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     3001 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3061 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3121 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3181 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3241 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3301 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tatcgcggta tcattgcagc
     3361 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3421 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3481 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3541 atttaaaagg atctaggtga agatcctttt tg
//
