LOCUS       sequence-435990-        7055 bp    DNA     circular SYN 15-APR-2025
DEFINITION  A plasmid encoding a photodegradable hydrogel-forming protein 
            PhoCoil.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7055)
  TITLE     PhoCoil: An Injectable and Photodegradable Single-component 
            Recombinant Protein Hydrogel for Localized Therapeutic Cell Delivery
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7055)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: PhoCoil
FEATURES             Location/Qualifiers
     source          1..7055
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     85..106
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        269..373
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(374..442,443..1234)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(592..611)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1405..1993
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1894..1913
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2147..2164
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2179..2318
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2244..2263)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2404..2426
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2420..2611)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3420..4502)
                     /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     4464..4483
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4503..4580)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4786..4805
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4889..4908
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4889..4907
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4908..4932
                     /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)."
     RBS             4947..4969
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4989..5006
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5466..6191
                     /codon_start=1
                     /product="photocleavable protein that separates into two 
                     fragments upon exposure to violet light (Zhang et al., 
                     2017)"
                     /label=PhoCl
                     /translation="VIPDYFKQSFPEGYSWERSMTYEDGGICIATNDITMEGDSFINKI
                     HFQGTNFPPNGPVMQKRTVGWEASTEKMYERDGVLKGDVKMKLLLKGGGHYRGDYRTTY
                     KVKQKPVKLPDCHFVDHRIEILSHDKDYNKVKLYEHAVAKTSTDSMDELYKGGSGGMVS
                     KGEETITSVIKPDMKNKLRMEGNVNGHAFVIEGEGSGKPFEGIQTIDLEVKEGAPLPFA
                     YDILTTAFHYGNRVFTKYPR"
     CDS             6648..6665
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6675..6692
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6745..6763)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6759..6806
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(6843..7055,1..243)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6930..6949)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
       61 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
      121 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
      181 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
      241 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
      301 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
      361 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
      421 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
      481 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
      541 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
      601 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
      661 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
      721 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
      781 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
      841 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
      901 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
      961 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     1021 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     1081 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     1141 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     1201 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     1261 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     1321 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     1381 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     1441 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     1501 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     1561 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     1621 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     1681 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     1741 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     1801 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     1861 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     1921 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     1981 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     2041 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     2101 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     2161 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2221 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     2281 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     2341 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     2401 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     2461 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     2521 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     2581 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     2641 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     2701 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     2761 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     2821 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     2881 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     2941 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     3001 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     3061 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     3121 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     3181 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     3241 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     3301 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     3361 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     3421 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3481 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     3541 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     3601 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     3661 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     3721 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     3781 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     3841 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     3901 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     3961 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     4021 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     4081 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     4141 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     4201 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     4261 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     4321 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     4381 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     4441 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     4501 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     4561 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     4621 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     4681 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     4741 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     4801 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     4861 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     4921 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgc
     4981 gtggcagcca ccatcaccat caccatgagt ttggccgcgg agattcagta gatggttcgg
     5041 gctctggatc aggttcgggt tcgggtagtg gggccccaca gatgcttcgc gagctccaag
     5101 agacaaacgc ggccctgcaa gacgtacgtg agcttctccg ccaggcagta aaagaaatta
     5161 catttctgaa gaatactgtt atggaaagcg acgcctcagg ttcgggttct gggtcaggca
     5221 gcggctccgg ctcattggat gggtcgccag caggtagtcc aacgagtacc gaggaaggta
     5281 cctcagaaag cgccactcca gaatcaggtc caggtacaag cactgagcct tcggaagggt
     5341 ctgcccccgg ctccccagcg ggcagtccca cgtcaacaga ggaaggcacg agtactgagc
     5401 cttcagaagg gtcagctcca gggacatcaa cggaacctag tgaaggcagc gccaaactgg
     5461 aattcgtgat ccctgactac ttcaagcaga gcttccccga gggctacagc tgggagcgca
     5521 gcatgaccta cgaggacggc ggcatctgca tcgccaccaa cgacatcaca atggaggggg
     5581 acagcttcat caacaagatc cacttccagg gcacgaactt cccccccaac ggccccgtga
     5641 tgcagaagag gaccgtgggc tgggaggcca gcaccgagaa gatgtacgag cgcgacggcg
     5701 tgctgaaggg cgacgtgaag atgaagctgc tgctgaaggg cggcggccac tatcgcggcg
     5761 actaccgcac cacctacaag gtcaagcaga agcccgtaaa gctgcccgac tgccacttcg
     5821 tggaccaccg catcgagatc ctgagccacg acaaggacta caacaaggtg aagctgtacg
     5881 agcacgccgt ggccaagact tccaccgaca gcatggacga gctgtacaag ggtggcagcg
     5941 gtggcatggt gagcaagggc gaggagacca ttacaagcgt gatcaagcct gacatgaaga
     6001 acaagctgcg catggagggc aacgtgaacg gccacgcctt cgtgatcgag ggcgagggca
     6061 gcggcaagcc cttcgagggc atccagacga ttgatttgga ggtgaaggag ggcgccccgc
     6121 tgcccttcgc ctacgacatc ctgaccaccg ccttccacta cggcaaccgc gtgttcacca
     6181 agtacccacg gggcggcggc caattgccgg gaacatcaga atctgctaca ccagaatcgg
     6241 gccctgggtc agaaccggcc acttcgggtt cagaaacacc tggcagtgaa cctgctacta
     6301 gtggctcgga aaccccaggg tccccagccg gttcccctac ctcgacagaa gagggtacat
     6361 cggagtccgc gacacctgaa tcagggccag gaacaagcac agaaccgtcg gaagggtctg
     6421 cccctctgga tggaagcggt tcaggctcgg gttcgggtag cggttcgggc gcaccccaga
     6481 tgttgcgcga gcttcaagag actaacgcag ccttacaaga cgtacgcgag ctgctgcgcc
     6541 aggccgtgaa agaaattacc ttcttaaaga atacggttat ggaaagtgac gcctcaggga
     6601 gtggtagtgg ttcgggcagc gggtcaggct ctggccgcgg cgattcacac catcaccatc
     6661 accattaact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     6721 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     6781 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     6841 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     6901 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     6961 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     7021 tagtgcttta cggcacctcg accccaaaaa acttg
//
