LOCUS       sequence-198213-        7733 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7733)
  TITLE     Craig lab unpublished plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7733)
  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            "Unpublished"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: pET15b/EYFP-GgVcl 884-1066-ECFP FRET
FEATURES             Location/Qualifiers
     source          1..7733
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(213..260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     256..274
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(join(330..1043,1044..1046,1047..1049))
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1644..2354,2355..2357,2358..2360))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(2364..2381)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2391..2408)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2427..2449
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2464..2488
                     /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)."
     primer_bind     complement(2488..2507)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2489..2507)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2591..2610)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2816..2893
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2894..3976
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2913..2932)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5288..5479
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5473..5495)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5581..5720
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5636..5655
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5735..5752)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5906..6494)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5986..6005)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6665..7456,7457..7525))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7288..7307
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7526..7630)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7698..7716
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
      241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
      301 cgggctttgt tagcagccgg atcctcgagt tacttgtaca gctcgtccat gccgagagtg
      361 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct
      421 ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg
      481 ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg
      541 tggcggatct tgaagttggc cttgatgccg ttcttctgct tgtcggcggt gatatagacg
      601 ttgtggctga tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag
      661 tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg
      721 cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg
      781 ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac
      841 tgcacgcccc aggtcagggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg
      901 gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac
      961 acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg
     1021 gtgaacagct cctcgccctt gctcaccatg ctagcctgat accatggggt ctttctgacc
     1081 cagcgcagag tgaatccggc atctgttctt atcttaatgg atgctgcttc agcttctctc
     1141 acagtttcct tcacagactg catgaggttc tgggcgttat gaaccaacat ctcagttgcc
     1201 tgttctgatt cttcatcgct gatgttagtc ctgcccagca tggtagcttt cactgtggaa
     1261 agaattttga gctgcgtgct gatggttggg attcgctcac agacctgtaa gaggtttgtt
     1321 ctaatgcgtt tatcagtaca ttgcttggcc acctctttgg ccaatcgagt gacttcatcc
     1381 gatgccttag caatatcttt tgcacactgg atgagggcac gcttgtttcc gctgcctcct
     1441 cgcaccaggc gtgacatctc agccatgagc agcgccattc gtttagcagc agcaatgatg
     1501 tcgttaccct tgctagacca tttccgagcc tcgtcatgca actgcctagc agccatcatc
     1561 atgggctgat taatagcttc tcctgctttc tgctctggga actccatatg ggcggcggcg
     1621 gggcaggggg ggccggaatt cgacttgtac agctcgtcca tgccgagagt gatcccggcg
     1681 gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc tttgctcagg
     1741 gcggactggt agctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg
     1801 gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc
     1861 ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg
     1921 ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc
     1981 ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg
     2041 tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg
     2101 gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc gggcgaagca ctgcaggccg
     2161 tagccgaagg tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg
     2221 aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac
     2281 ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc
     2341 tcctcgccct tgctcaccat atggctgccg cgcggcacca ggccgctgct gtgatgatga
     2401 tgatgatggc tgctgcccat gctatatctc cttcttaaag ttaaacaaaa ttatttctag
     2461 aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc
     2521 gagatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
     2581 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     2641 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     2701 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     2761 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac
     2821 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     2881 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     2941 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     3001 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     3061 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     3121 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     3181 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     3241 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     3301 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     3361 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     3421 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     3481 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     3541 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     3601 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     3661 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac
     3721 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
     3781 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     3841 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     3901 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     3961 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg
     4021 gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
     4081 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg
     4141 tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga
     4201 tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg
     4261 gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg
     4321 cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga
     4381 ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg
     4441 tagatgacga ccatcaggga cagcttcaag gatcgctcgc ggctcttacc agcctaactt
     4501 cgatcattgg accgctgatc gtcacggcga tttatgccgc ctcggcgagc acatggaacg
     4561 ggttggcatg gattgtaggc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg
     4621 gtgcatggag ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt
     4681 caccactcca agaattggag ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa
     4741 cccttggcag aacatatcca tcgcgtccgc catctccagc agccgcacgc ggcgcatctc
     4801 gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg
     4861 gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac
     4921 gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg
     4981 tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg
     5041 gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa
     5101 gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg
     5161 tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag
     5221 catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga
     5281 ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca
     5341 gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg
     5401 tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga
     5461 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     5521 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     5581 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     5641 tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa
     5701 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5761 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5821 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5881 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5941 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     6001 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6061 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6121 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6181 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6241 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6301 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     6361 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6421 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6481 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6541 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6601 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6661 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6721 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6781 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6841 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6901 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6961 gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     7021 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     7081 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7141 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7201 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7261 gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag
     7321 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7381 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7441 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7501 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7561 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7621 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     7681 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaa
//
