LOCUS       sequence-442992-        4331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used in phosphoprotein synthesis. Replace the sfGFP with gene of 
            interest (GOI). Add TAG amber codon(s) at the position(s) in GOI 
            where phosphoserine incorporation is desired..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4331)
  TITLE     Rinehart Lab Phosphoprotein synthesis components
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4331)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pCRT7 NT Topo tetR/pLtetO Amp‐WT sfGFP
FEATURES             Location/Qualifiers
     source          1..4331
                     /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"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(940..1563)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     1471..1490
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    1612..1628
                     /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)."
     protein_bind    1635..1651
                     /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        join(1660..1678,1679..1684,1685..1701,1702..1707,
                     1708..1733)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1660..1678
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1685..1703
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1743..1754
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(1761..1763,1764..2474)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2481..2498
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2557..2575)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2571..2618
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2715..3170
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2802..2821)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3012..3033
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3196..3300
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3301..3369,3370..4161)
                     /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(3519..3538)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
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 gaagccaaag ggcccaaggc
      781 ccagtctttc gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcat
      841 ggggagaccc cacactacca tcggcgctac ggcgtttcac ttctgagttc ggcatggggt
      901 caggtgggac caccgcgcta ctgccgccag gcatctagat taagacccac tttcacattt
      961 aagttgtttt tctaatccgc atatgatcaa ttcaaggccg aataagaagg ctggctctgc
     1021 accttggtga tcaaataatt cgatagcttg tcgtaataat ggcggcatac tatcagtagt
     1081 aggtgtttcc ctttcttctt tagcgacttg atgctcttga tcttccaata cgcaacctaa
     1141 agtaaaatgc cccacagcgc tgagtgcata taatgcattc tctagtgaaa aaccttgttg
     1201 gcataaaaag gctaattgat tttcgagagt ttcatactgt ttttctgtag gccgtgtacc
     1261 taaatgtact tttgctccat cgcgatgact tagtaaagca catctaaaac ttttagcgtt
     1321 attacgtaaa aaatcttgcc agctttcccc ttctaaaggg caaaagtgag tatggtgcct
     1381 atctaacatc tcaatggcta aggcgtcgag caaagcccgc ttatttttta catgccaata
     1441 caatgtaggc tgctctacac ctagcttctg ggcgagttta cgggttgtta aaccttcgat
     1501 tccgacctca ttaagcagct ctaatgcgct gttaatcact ttacttttat ctaatctgga
     1561 catgtcgaat atagacgtcg gtcagtgcgt cctgctgatg tgctcagtat cttgttatcc
     1621 gctcacaatg tcaattgtta tccgctcaca attctcgagt ccctatcagt gatagagatt
     1681 gacatcccta tcagtgatag agatactgag cacatcagca ggacgcactg accgaattca
     1741 ttaaagagga gaaaggtacc atgagcaagg gcgaagaact gtttacgggc gtggtgccga
     1801 ttctggtgga actggatggt gatgtcaatg gtcacaaatt cagcgtgcgc ggcgaaggtg
     1861 aaggcgatgc aaccaatggt aaactgacgc tgaagtttat ttgcaccacg ggtaaactgc
     1921 cggttccgtg gccgaccctg gtcaccacgc tgacgtatgg tgttcagtgt ttcagtcgtt
     1981 acccggatca catgaaacgc cacgactttt tcaagtccgc gatgccggaa ggttatgtcc
     2041 aagaacgtac catctcattt aaagatgacg gcacctacaa aacgcgcgcc gaagtgaaat
     2101 tcgaaggtga tacgctggtt aaccgtattg aactgaaagg catcgatttt aaggaagacg
     2161 gtaatattct gggccataaa ctggaatata acttcaattc gcacaacgtg tacatcaccg
     2221 cagataagca gaagaacggt atcaaggcta acttcaagat ccgccataat gtggaagatg
     2281 gcagcgttca actggccgac cactatcagc aaaacacccc gattggtgat ggcccggtcc
     2341 tgctgccgga caatcattac ctgagcacgc agtctgtgct gagtaaagat ccgaacgaaa
     2401 agcgtgacca catggtcctg ctggaattcg tgaccgcggc cggcatcacg cacggtatgg
     2461 acgaactgta taaaggctca catcatcatc atcatcattg aaagcttgat ccggctgcta
     2521 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     2581 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     2641 gatctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     2701 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2761 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2821 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2881 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2941 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3001 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3061 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3121 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     3181 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3241 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3301 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3361 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3421 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3481 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3541 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3601 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3661 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3721 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3781 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3841 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3901 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3961 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4021 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4081 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4141 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4201 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4261 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4321 aaaggatctt c
//
