LOCUS       pMIA10.53_Clover        4616 bp DNA     circular SYN 05-JAN-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4616)
  AUTHORS   Matias
  TITLE     Direct Submission
  JOURNAL   Exported Jan 5, 2024 from SnapGene 7.1.0
            https://www.snapgene.com
COMMENT     Sequence confirmed with nanopore 
            N2314441_30-920089658_Pacbio_2023-09-13
FEATURES             Location/Qualifiers
     source          1..4616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..48)
                     /label=pGEX3
     primer_bind     220..237
                     /label=CF 2nd M HindIII insu R
     primer_bind     220..237
                     /label=NotI R
     primer_bind     290..307
                     /label=BamHI Stop R
     primer_bind     359..377
                     /label=U-19mer Primer
     primer_bind     359..375
                     /label=M13F-pUC
     primer_bind     360..377
                     /label=loxP Bsd R
     primer_bind     378..395
                     /label=M13-FP
     primer_bind     379..395
                     /label=M13F
     protein_bind    join(413..434,435..436,437..458)
                     /label=BxbI attB - CT
                     /label=BxbI attB
                     /bound_moiety="BxbI integrase"
                     /note="attB site for the BxbI integrase (Ghosh, P et.al.; 
                     2003)
                     central di-nucleotide mutated to cT
                     "
     regulatory      465..474
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(471..473,474..476,477..1187)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(479..496)
                     /label=pEGFP-RP
     primer_bind     complement(516..537)
                     /label=EGFP-N
     primer_bind     complement(516..532)
                     /label=pEGFP_N
     primer_bind     complement(522..537)
                     /label=EGFP-NR
     primer_bind     complement(536..553)
                     /label=clover integration R
     primer_bind     complement(538..556)
                     /label=clover screen R
     primer_bind     complement(539..556)
                     /label=kcnj-gpf I R
     primer_bind     complement(540..557)
                     /label=GFP-N R
     primer_bind     607..628
                     /label=GFP R F
     primer_bind     complement(829..850)
                     /label=GFP R R
     primer_bind     966..983
                     /label=Clover C F II
     primer_bind     1010..1027
                     /label=Clover C F
     primer_bind     1011..1029
                     /label=GFP-C F
     primer_bind     1124..1145
                     /label=EGFP-C
     primer_bind     1172..1189
                     /label=clover screen F
     primer_bind     complement(1195..1212)
                     /label=BamHI Stop R
     misc_feature    join(1247..1821,1822..1824,1825..1833)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     1737..1754
                     /label=IRES F
     CDS             1834..2433
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1981..1998)
                     /label=CF Puro R
     primer_bind     1982..1999
                     /label=puro F
     primer_bind     complement(2328..2345)
                     /label=puro screen R
     primer_bind     2350..2363
                     /label=puroend_fwd
     protein_bind    join(2437..2458,2459..2460,2461..2482)
                     /label=BxbI attB - GT
                     /label=BxbI attB
                     /bound_moiety="BxbI integrase"
                     /note="attB site for the BxbI integrase (Ghosh, P et.al.; 
                     2003)"
     primer_bind     complement(2537..2556)
                     /label=RVprimer4
     rep_origin      complement(2797..3385)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2824..2841)
                     /label=mRosa EcoRV 1 F
     primer_bind     3216..3233
                     /label=Amp-pUC R
     CDS             complement(join(3556..4347,4348..4416))
                     /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     complement(3767..3784)
                     /label=Amp-pUC F
     promoter        complement(4417..4521)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4502..4519
                     /label=mRosa 3' HindIII R
     primer_bind     4520..4537
                     /label=FLP insert F
     primer_bind     4530..4547
                     /label=pQE-F
     primer_bind     4531..4547
                     /label=QE Promoter
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gagctagcta taggccggct
      421 tgtcgacgac ggcgctctcc gtcgtcagga tcatccggac cggtgccacc atggtgagca
      481 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
      541 acggccacaa gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac ggcaagctga
      601 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
      661 ccttcggcta cggcgtggcc tgcttcagcc gctaccccga ccacatgaag cagcacgact
      721 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctct ttcaaggacg
      781 acggtaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
      841 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
      901 acaacttcaa cagccacaac gtctatatca cggccgacaa gcagaagaac ggcatcaagg
      961 ctaacttcaa gatccgccac aacgttgagg acggcagcgt gcagctcgcc gaccactacc
     1021 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagcc
     1081 atcagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1141 tcgtgaccgc cgccgggatt acacatggca tggacgagct gtacaagtaa taactggcgt
     1201 aatagcgaag aggcccgcac cgatcgccct tcccaacagt gaattccccc tctccctccc
     1261 ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata
     1321 tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg
     1381 tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt
     1441 tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag
     1501 cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc
     1561 cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga
     1621 tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg
     1681 cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat
     1741 gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc
     1801 tttgaaaaac acgatgataa tatggccaca accatgaccg agtacaagcc cacggtgcgc
     1861 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     1921 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     1981 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     2041 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     2101 gagattggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     2161 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     2221 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     2281 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgcccca caacctcccc
     2341 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     2401 acctggtgca tgacccgcaa gcccggtgcc taataaggcc ggcttgtcga cgacggcggt
     2461 ctccgtcgtc aggatcatcc ggttaattaa ccgatgccct tgagagcctt caacccagtc
     2521 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     2581 atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc tcactgactc
     2641 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2701 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2761 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2821 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2881 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2941 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3001 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3061 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3121 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3181 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3241 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3301 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3361 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3421 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3481 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3541 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3601 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3661 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3721 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3781 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3841 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3901 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3961 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4021 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4081 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4141 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4201 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4261 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4321 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4381 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4441 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4501 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     4561 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
