LOCUS       pmetalfa-mcherry        6175 bp    DNA     circular SYN 11-JUN-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6175)
  AUTHORS   Alessandra Affolter Lab
  TITLE     Direct Submission
  JOURNAL   Exported Jun 11, 2025 from SnapGene 8.1.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6175
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     primer_bind     135..152
                     /label=pcdnafor
     primer_bind     223..263
                     /label=testcmvpromfor
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     726..745
                     /label=EGFP-N-for
     primer_bind     769..789
                     /label=CMVfor
     primer_bind     769..789
                     /label=CMV-for
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    841..859
                     /label=tet operator
     primer_bind     985..1006
                     /label=metalfa_for
     primer_bind     993..1009
                     /label=alfaformito_for
     CDS             994..1038
                     /codon_start=1
                     /label=ALFA
                     /note="ALFA"
                     /translation="PSRLEEELRRRLTEP"
     primer_bind     994..1006
                     /label=testalfahorh2b_for
     primer_bind     complement(1022..1046)
                     /label=lafaforh2b_rev2
     primer_bind     complement(1023..1043)
                     /label=alfaformito_rev
     primer_bind     complement(1024..1041)
                     /label=alfaforh2b_rev
     primer_bind     1046..1071
                     /label=mcherry5pletm1_for
     primer_bind     1052..1074
                     /label=testtfprab5cmvfor
     CDS             1054..1761
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFSEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     1054..1074
                     /label=mTFPmito_for
     primer_bind     complement(1733..1761)
                     /label=mtfprab5f.REV
     primer_bind     complement(1733..1761)
                     /label=mtfprab5f.REVa
     primer_bind     complement(1740..1760)
                     /label=testtfprabcmvrev
     primer_bind     complement(1743..1762)
                     /label=mTFPmito_rev
     primer_bind     1767..1785
                     /label=h2bnterm_for
     misc_feature    1768..2148
                     /label=HIST1H2BJ
     primer_bind     complement(2126..2145)
                     /label=h2bnterm_rev
     primer_bind     complement(2186..2203)
                     /label=BGH-rev
     polyA_signal    2192..2416
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2462..2890
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2904..3233
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3084..3219
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3281..3328
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3347..3721
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     primer_bind     complement(3553..3571)
                     /label=testtfprabcmvrev
     polyA_signal    3851..3972
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3851..3868)
                     /label=EGFP-C-Rev
     primer_bind     complement(4021..4037)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4045..4061
                     /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(4069..4075,4076..4093,4094..4099))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4114..4135
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4423..5008)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5179..5970,5971..6039))
                     /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"
     promoter        complement(6040..6144)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggaacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttgatcgca atgccatcac gtttggaaga ggaactgaga
     1021 cgccgcttaa ctgaacctgg ccggatcgcc accatggtga gcaagggcga ggaggataac
     1081 atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc
     1141 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     1201 aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag
     1261 ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag
     1321 ctgtccttct ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg
     1381 gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg
     1441 cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag
     1501 gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg
     1561 ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag
     1621 aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac
     1681 aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc
     1741 ggcatggacg agctgtacaa ggatatcatg ccagagccag cgaagtctgc tcccgccccg
     1801 aaaaagggct ccaagaaggc ggtgactaag gcgcagaaga aaggcggcaa gaagcgcaag
     1861 cgcagccgca aggagagcta ttccatctat gtgtacaagg ttctgaagca ggtccaccct
     1921 gacaccggca tttcgtccaa ggccatgggc atcatgaatt cgtttgtgaa cgacattttc
     1981 gagcgcatcg caggtgaggc ttcccgcctg gcgcattaca acaagcgctc gaccatcacc
     2041 tccagggaga tccagacggc cgtgcgcctg ctgctgcctg gggagttggc caagcacgcc
     2101 gtgtccgagg gtactaaggc catcaccaag tacaccagcg ctaagtgact cgagtctaga
     2161 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2221 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2281 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2341 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2401 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     2461 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2521 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2581 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2641 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2701 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2761 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2821 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2881 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     2941 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     3001 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3061 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3121 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     3181 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     3241 cgggagcttg tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc
     3301 atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac
     3361 cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga
     3421 ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga
     3481 cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg
     3541 ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa
     3601 cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga
     3661 gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg
     3721 acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     3781 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     3841 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3901 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3961 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     4021 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4081 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4141 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4201 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4261 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4321 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4381 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4441 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4501 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4561 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4621 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4681 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4741 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4801 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4861 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4921 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ttttttgttt gcaagcagca
     4981 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5041 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     5101 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5161 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     5221 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     5281 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     5341 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     5401 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     5461 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     5521 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     5581 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     5641 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     5701 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     5761 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     5821 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5881 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5941 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     6001 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     6061 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     6121 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
